Promoters exhibiting endothelial cell specificity and methods of using same

ABSTRACT

An isolated polynucleotide functional as a promoter in eukaryotic cells is disclosed. The isolated polynucleotide includes an endothelial specific enhancer element as detailed herein. Further disclosed is a method of expressing a nucleic acid sequence of interest in endothelial cells.

FIELD AND BACKGROUND OF THE INVENTION

[0001] The present invention relates to isolated polynucleotidesequences exhibiting endothelial cell specific promoter activity, andmethods of use thereof and, more particularly, to amodified-preproendothelin-1 (PPE-1) promoter which exhibits increasedactivity and specificity in endothelial cells. The invention furtherrelates to modifications of the PPE promoter which enhance itsexpression in response to physiological conditions including hypoxia andangiogenesis.

[0002] Gene therapy is an emerging modality for treating inherited andacquired human diseases. Great efforts are directed towards developingmethods for gene therapy of cancer, cardiovascular and peripheralvascular diseases, but there is still a major obstacle in effective andspecific gene delivery. In general, the main limiting factor of genetherapy with a gene of interest using a recombinant viral vector as ashuttle is the ability to specifically direct the gene of interest tothe target tissue. Adenoviruses that are used for this purpose arecapable of infecting a large variety of cells with different affinities.Indeed, using a non-tissue specific promoter induces up to 95% of theexpression of the gene of interest in the liver; hence regulation ofexpression is highly required. Further, it is currently infeasible todifferentiate between normal vascular endothelia and developing vascularendothelia in a growing tumor when targeting a gene.

[0003] In both cancer development and vascular diseases, angiogenesis,the creation of new vessels, plays a central role. Hence, regulation ofthis process by gene therapy to the vascular endothelium can betremendously important in inducing targeted therapy for these diseases.

[0004] High efficiency of the human preproendothelin-1 (PPE-4),delivered by retroviral vector, was obtained in endothelial cells (EC)in-vitro, and in transgenic animal models. However, the prior art doesnot teach use of this promoter for in-vivo gene therapy. The human PPE-1lacks regulatory elements found in PPE-1 genes of other animals, mostnotably the mouse.

[0005] U.S. Pat. No. 5,747,340 teaches use of the murine PPE-1 promoterand portions thereof. However, this patent contains no hint orsuggestion that an endothelial-specific enhancer can be employed toincrease the level of expression achieved with the PPE promoter whilepreserving endothelial specificity. Further, this patent does not teachthat the PPE-1 promoter is induced to higher levels of transcriptionunder hypoxic conditions.

[0006] There is thus a widely recognized need for, and it would behighly advantageous to have, an improved endothelial cell specificpromoter, methods of use thereof and cells transformed therewith devoidof the above limitations. The disclosed invention is expected todemonstrate added utility in treatment of cardiovascular disease, cancerand wound healing relative to previously known configurations.

SUMMARY OF THE INVENTION

[0007] According to one aspect of the present invention there isprovided an isolated polynucleotide functional as a promoter ineukaryotic cells. The isolated polynucleotide includes an enhancerelement including at least two copies of the sequence set forth in SEQID NO:6.

[0008] According to another aspect of the present invention there isprovided a method of expressing a nucleic acid sequence of interest,encoding an active RNA molecule or a protein such as an enzyme, reportermolecule and the like in endothelial cells. The method includesadministering to a subject a construct which includes the nucleic acidsequence of interest positioned under the regulatory control of apromoter functional in eukaryotic cells. The construct further includesan enhancer element including at least one copy of the sequence setforth in SEQ ID NO:6.

[0009] According to yet another aspect of the present invention there isprovided a method of regulating angiogenesis in a tissue. The methodincludes administering a nucleic acid construct including: (a) anendothelial cell specific promoter; (b) at least one copy of a hypoxiaresponse element set forth in SEQ ID NO:5; and (c) a nucleic acidsequence encoding an angiogenesis regulator, the nucleic acid sequencebeing under regulatory control of the promoter and the hypoxia responseelement.

[0010] According to still another aspect of the present invention thereis provided an isolated polynucleotide functional as a promoter ineukaryotic cells. The isolated polynucleotide includes an enhancerelement including the sequence set forth in SEQ ID NO: 7.

[0011] According to an additional aspect of the present invention thereis provided a method of regulating angiogenesis in a tissue. The methodincludes administering a nucleic acid construct including: (a) anendothelial cell specific promoter; (b) an enhancer element includingthe sequence set forth in SEQ ID NO: 7; (c) at least one copy of ahypoxia response element set forth in SEQ ID NO:5; and (d) a nucleicacid sequence encoding an angiogenesis regulator, the nucleic acidsequence being under regulatory control of the promoter, the enhancerelement and the hypoxia response element.

[0012] According to still a further aspect of the present inventionthere is provided isolated polynucleotide functional as a promoter ineukaryotic cells, the isolated polynucleotide includes an enhancerelement including at least one copy of the sequence set forth in SEQ IDNO:8.

[0013] According to still a further aspect of the present inventionthere is provided a method of expressing a nucleic acid sequence ofinterest in endothelial cells, the method includes administering to asubject a construct, the construct includes the nucleic acid sequence ofinterest positioned under the regulatory control of a promoterfunctional in eukaryotic cells, and an enhancer element including atleast one copy of the sequence set forth in SEQ ID NO:8.

[0014] According to still another further aspect of the presentinvention there is provided isolated polynucleotide functional as apromoter in eukaryotic cells, the isolated polynucleotide includes anenhancer element including the sequence set forth in SEQ ID NO: 8.

[0015] According to further features in preferred embodiments of theinvention described below, the enhancer element includes three copies ofthe sequence set forth in SEQ ID NO:6.

[0016] According to still further features in preferred embodiments ofthe invention the at least two copies of the sequence set forth in SEQID NO:6 are contiguous.

[0017] According to still further features in preferred embodiments ofthe invention the isolated polynucleotide further includes anendothelial specific promoter element.

[0018] According to still further features in preferred embodiments ofthe invention the endothelial specific promoter element includes atleast one copy of the PPE-1 promoter.

[0019] According to still further features in preferred embodiments ofthe invention the isolated polynucleotide further includes a hypoxiaresponse element.

[0020] According to still further features in preferred embodiments ofthe invention the hypoxia response element includes at least one copy ofthe sequence set forth in SEQ ID NO: 5.

[0021] According to still further features in preferred embodiments ofthe invention the enhancer element is as set forth in SEQ ID NO: 7.

[0022] According to still further features in preferred embodiments ofthe invention there is provided a nucleic acid construct including aclaimed isolated polynucleotide and a nucleic acid sequence of interest,the nucleic acid sequence of interest being under regulatory control ofthe isolated polynucleotide.

[0023] According to still further features in preferred embodiments ofthe invention the nucleic acid sequence of interest is selected from thegroup consisting of VEGF, p55 and PDGF-BB.

[0024] According to still further features in preferred embodiments ofthe invention there is provided a mammalian cell transformed with aclaimed isolated polynucleotide.

[0025] According to still further features in preferred embodiments ofthe invention the promoter exhibits endothelial cell specificity.

[0026] According to still further features in preferred embodiments ofthe invention the promoter is the PPE-1 promoter as set forth in SEQ IDNO: 1.

[0027] According to still further features in preferred embodiments ofthe invention administering is effected by a method selected from thegroup consisting of: (i) systemic in-vivo administration; (ii) ex-vivoadministration to cells removed from a body of a subject and subsequentreintroduction of the cells into the body of the subject; and (iii)local in-vivo administration.

[0028] According to still further features in preferred embodiments ofthe invention the nucleic acid construct further includes an enhancerelement including at least two copies of the sequence set forth in SEQID NO:6.

[0029] According to still further features in preferred embodiments ofthe invention the endothelial cell specific promoter includes at leastone copy of the PPE-1 promoter.

[0030] According to still further features in preferred embodiments ofthe invention there is provided a nucleic acid construct including aclaimed isolated polynucleotide and a nucleic acid sequence of interest,the nucleic acid sequence of interest being under regulatory control ofthe isolated polynucleotide.

[0031] According to still further features in the described preferredembodiments the enhancer element further includes at least one copy ofthe sequence set forth in SEQ ID NO:6.

[0032] According to still further features in the described preferredembodiments the enhancer element includes one copy of the sequence setforth in SEQ ID NO:8 and at least two copies of the sequence set forthin SEQ ID NO:6.

[0033] According to still further features in the described preferredembodiments the enhancer element further includes at least one copy ofthe sequence set forth in SEQ ID NO:6.

[0034] According to still further features in the described preferredembodiments the at least one copy includes two copies.

[0035] According to still further features in the described preferredembodiments the nucleic acid construct further includes an enhancerelement including at least one copy of the sequence set forth in SEQ IDNO:8.

[0036] According to yet another further aspect of the present inventionthere is provided method of regulating angiogenesis in a tissue, themethod comprising administering a nucleic acid construct including: (a)an endothelial cell specific promoter; (b) an enhancer element includingat least one copy of the sequence set forth in SEQ ID NO:8; and (c) anucleic acid sequence encoding an angiogenesis regulator, the nucleicacid sequence being under regulatory control of the promoter and theenhancer element.

[0037] According to still further features in the described preferredembodiments the enhancer element further includes at least one copy ofthe sequence set forth in SEQ ID NO:6.

[0038] According to still further features in the described preferredembodiments the enhancer element includes one copy of the sequence setforth in SEQ ID NO:8 and at least two copies of the sequence set forthin SEQ ID NO:6.

[0039] The present invention successfully addresses the shortcomings ofthe presently known configurations by providing improved isolatedpolynucleotide sequences with endothelial cell specificity, and methodsof use thereof. The improvements in the sequence make feasible methodsof treating a variety of diseases, disorders and conditions which werepreviously considered infeasible. Specifically, the improvements relateto increased specificity to endothelial cells, increased levels ofexpression of a sequence of interest and enhanced induction byconditions including ischemia and angiogenesis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

[0041] In the drawings:

[0042]FIG. 1 is a histogram illustrating the effect of the enhancerelement of the present invention on Luciferase expression in both bovineand human endothelial cell lines using the B2B cell line as anon-endothelial control.

[0043]FIG. 2 is a histogram illustrating endothelial specificity of apromoter of the present invention in an adenoviral vector on Luciferaseexpression in various cell lines.

[0044]FIGS. 3A and 3B are photomicrographs illustrating GFP expressionunder the control of Ad5PPE-1-3X of the present invention and an Ad5CMVcontrol construct in the BAEC cell line.

[0045]FIG. 4 is histogram of % apoptosis induced by pACPPE-1-3Xp55,pACPPE-1-3XLuciferase and pCCMVp55 in endothelial and non-endothelialcells.

[0046]FIG. 5 is a histogram illustrating the effect of introducing anenhancer element according to the present invention into a promoterconstruct on hypoxia response.

[0047]FIG. 6 is a histogram illustrating the effect of introducing anenhancer element according to the present invention into a promoter ofan adenovector construct on hypoxia response.

[0048]FIG. 7 is a histogram illustrating the effect of introducing anenhancer element according to the present invention into a promoter onlevels of expression in bovine and human endothelial cell lines.

[0049]FIG. 8 is a histogram illustrating levels of expression of areporter gene observed in various organs after injection of anadenoviral construct either an endothelial promoter (PPE-1) or a control(CMV) promoter;

[0050] FIGS. 9A-B are two photomicrographs illustrating cellularexpression of an Ad5CMVGFP construct (FIG. 9a) and an Ad5PPE-1-GFPconstruct (FIG. 9b) in liver tissue of mice injected with theconstructs.

[0051]FIG. 10 is a histogram illustrating the effect of introducing anenhancer element according to the present invention into a promoter onlevels of expression in endothelial and non-endothelial cell lines.

[0052]FIG. 11 is a histogram illustrating the effect of introducing anenhancer element according to the present invention into a promoter onlevels of expression in endothelial and non-endothelial cell lines.

[0053] FIGS. 12A-C are photomicrographs illustrating GFP expression inAd5PPE-1-3XGFP transduced cells, Ad5PPE-IGFP transduced cells andAd5CMVGFP transduced cells respectively.

[0054] FIGS. 13A-B illustrate GFP expression in SMC transduced by moi-1of Ad5PPE-1-3XGFP and Ad5 CMVGFP respectively.

[0055] FIGS. 14A-B show results of an experiment similar to that ofFIGS. 13A-B conducted in HeLa cells.

[0056] FIGS. 15A-B show results of an experiment similar to that ofFIGS. 13A-B conducted in HepG2 cells.

[0057] FIGS. 16A-B show results of an experiment similar to that ofFIGS. 13a-b conducted in NSF cells.

[0058] FIGS. 17A-B are photomicrographs illustrating GFP expression inendothelial cells lining a blood vessel of mice injected with theAd5PPE-1GFP and the Ad5PPE-1-3XGFP constructs respectively.

[0059] FIGS. 18A-C are photomicrographs illustrating results from kidneytissue of injected mice. Ad5CMVGFP injected mice (FIG. 18A), Ad5PPE-1GFP(FIG. 18B; slightly higher GFP expression is visible in the blood vesselwall; indicated by arrow) and Ad5PPE-1-3XGFP (FIG. 18C).

[0060] FIGS. 19A-C illustrate experiments similar to those depicted inFIGS. 18A-C, conducted on sections of spleen tissue.

[0061] FIGS. 20A-D and 20C′-D′ illustrate GFP expression in metastaticlungs of control mice injected with Saline (FIG. 20A), mice injectedwith Ad5CMVGFP (FIG. 20B), mice injected with Ad5PPE-1GFP (FIG. 20C) andmice injected with Ad5PPE-1-3XGFP (FIG. 20D). Anti Cd31 immunostaining(FIGS. 20C′ to 20D′) confirm the co-localization of the GFP expressionand CD31 expression in each metastatic tissue.

[0062]FIG. 21 is a histogram illustrating that Luciferase activity(light units/μg protein) in BAEC transfected by a plasmid containing themurine PPE-1 promoter is significantly higher when transfected cellswere incubated under hypoxic conditions.

[0063]FIG. 22 is a histogram as in FIG. 21, except that Ad5PPE-1Luc andAd5CMVLuc were employed.

[0064]FIG. 23 is a series of histograms as in FIG. 22 showing theeffects of hypoxia in different cell lines.

[0065]FIG. 24 is a histogram illustrating the effect of the 3X sequenceof the present invention on the PPE-1 hypoxia response in BAEC cells.Cells were transduced by Ad5PPE-1Luc and Ad5PPE-1-3XLuc.

[0066]FIG. 25 is a histogram showing levels of Luciferase expression inPPE1-Luc transgenic mice following femoral artery ligation.

[0067] FIGS. 26A-B are plasmid maps of constructs employed inconjunction with the present invention.

[0068] FIGS. 27A-D are a series of ultrasound images of ligated limbs ofj representative animals from the different treatment groups, 21 daysfollowing ligation. FIG. 27A Control, Ad5CMVLuc. Treated; FIG. 27BControl, saline treated; FIG. 27C Ad5PPE-3X-VEGF treated; FIG. 27DAd5CMV-VEGF treated.

[0069]FIG. 28 is a histogram illustrating Luciferase activity inproliferating and quiescent Bovine Aortic Endothelial Cells (BAEC)transduced with Ad5PPE-1Luc (open bars) and Ad5CMVLuc (black bars).

[0070]FIG. 29 is a histogram illustrating Luciferase activity in BAECtransduced with Ad5PPE-1Luc during normal proliferation, a quiescentstate and rapid proliferation following addition of VEGF.

[0071] FIGS. 30A-B are histograms illustrating Luciferase activity(light units/μg protein) in the (FIG. 30A) aortas and livers (FIG. 30B)of Ad5PPE-1Luc and Ad5CMVLuc normal injected C57BL/6 mice. Activitieswere determined 1 (n=13), 5 (n=34), 14 (n=32), 30 (n=20) and 90 (n=11)days post injection.

[0072] FIGS. 31A-B are histograms illustrating relative Luciferaseactivity (light units/μg protein) detected five (FIG. 31A) and fourteen(FIG. 31B) (n=10 for each time point) days post injection of Ad5PPE-1Luc(open bars) or Ad5CMVLuc (black bars) in normal injected BALB/C mice.Activity is expressed as percentage of total body Luciferase expressionof each animal.

[0073]FIG. 32 is a prior art image depicting an Aorta dissected fromApoE deficient mice colored by Sudan-IV. The thoracic aorta containsless red stained atherosclerotic lesion while the abdominal regionincludes many red stained atherosclerotic lesions. (Adapted from Imagingof Aortic atherosclerotic lesions by ¹²⁵I-HDL and ¹²⁵I-BSA. A. Shaish etal, Pathobiology—submitted for publication).

[0074]FIG. 33 is a histogram illustrating absolute Luciferase activity(light units/μg protein) detected 5 days post systemic injections ofAd5PPE-1Luc (open bars; n=12) or Ad5CMVLuc (black bars; n=12) to ApoEdeficient mice. Luciferase activity observed from the abdominal aortacontain high lesion levels and from the thoracic area (low lesionlevels).

[0075]FIG. 34 is a histogram illustrating absolute Luciferase activity(light units/μg protein) 5 days post systemic injections of Ad5PPE-1Luc(black bars) or Ad5CMVLuc (open bars) to healing wound C57BL/6 inducedmice.

[0076]FIG. 35 is a histogram illustrating Luciferase activity in normallung, metastatic lung and primary tumor of Lewis lung carcinoma-inducedmice. Lewis lung carcinoma was induced by D122-96 cells injection to thebacks for primary tumor model and to the footpad for the metastaticmodel. Luciferase activity was measured five days post-systemicinjection of Ad5PPE-1Luc (n=9; open bars) or Ad5CMVLuc (n=12; blackbars). Activity is expressed as light units/μg protein.

[0077] FIGS. 36A-D are photomicrographs illustrating GFP expression andtissue morphology in lungs and tumors of LLC bearing mice afterintra-tumoral injection of Ad5PPE-1GFP. Tissue was frozen in OCT andsectioned to 10 μm by cryostat. All pictures were taken in magnificationof 25×. FIG. 36A-GFP in angiogenic blood vessels of lung metastases;FIG. 36B—CD31 antibody immuno-staining of the section pictured in FIG.36aA; FIG. 36C—GFP expression in blood vessels of primary tumor; FIG.36D—phase contrast of the section of C illustrating blood vessels.

[0078]FIG. 37 is a histogram illustrating Luciferase expression innormal lung and metastatic lung of Lewis lung carcinoma-induced mice,injected with Ad5CMVLuc, Ad5PPE-1Luc and Ad5PPE-1-3X-Luc Lewis lungcarcinoma was induced by Dl 22-96 cells injected to the foot pad for themetastatic model. Luciferase activity was measured five dayspost-systemic injection of Ad5CMVLuc (n=7; black bars), Ad5PPE-1Luc(n=6; gray bars), or Ad5PPE-1-3XLuc (n=13; brown bars). Activity isexpressed as light units/μg protein.

[0079]FIG. 38 is a histogram illustrating Luciferase activity aspercentage of liver activity (where the liver is 100%), in normal lungand lung metastasis of Lewis lung carcinoma-induced mice injected withAd5CMV, Ad5PPE-1Luc and Ad5PPE-1(3X).

[0080] FIGS. 39A-B are photomicrographs illustrating co-localization ofGFP expression (FIG. 39A) and Cd31 immuno-staining (FIG. 39B) in micewith LLC lung metastases injected with Ad5PPE-1-3X-GFP.

[0081]FIG. 40 is a histogram illustrating Luciferase activity (lightunits/μg protein) in muscles (ischemic and normal) of PPE-1Luciferasetransgenic mice at two, five, ten and 18 days post femoral ligation andin control (non-ligated animals—day 0; n=8 for each group).

[0082]FIG. 41 is a histogram illustrating Luciferase activity (lightunits/μg protein) in the liver, lung and aorta in muscles (ischemic andnormal) of PPE-1Luciferase transgenic mice at five (n=6), ten (n=6) and18 (n=8) days post femoral ligation and in control (non ligatedanimals—day 0).

[0083]FIG. 42 is a histogram illustrating Luciferase activity, (lightunits/μg protein detected in the livers, lungs and primary tumors of LLCmice injected in primary tumors with Ad5CMVLuc (black bars) orAd5PPE-1Luc (open bars).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] The present invention is of an improved endothelial cell-specificpromoter which can be employed to reliably direct high-level expressionof a sequence of interest to endothelial cells and in particularendothelial cells participating in angiogenesis.

[0085] The principles and use of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

[0086] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement of thecomponents set forth in the following description or illustrated in theexamples and drawings. The invention is capable of other embodiments orof being practiced or carried out in various ways. Also, it is to beunderstood that the phraseology and terminology employed herein is forthe purpose of description and should not be regarded as limiting.

[0087] Although endothelial specific promoters have been previouslydescribed (e.g. U.S. Pat. No. 5,747,340) these promoters have typicallybeen inefficient at directing expression to endothelial cells or havenot been demonstrated as being specific to endothelia cells in-vivo.

[0088] Enhancer elements specific to endothelial cells have also beendescribed. Bu et al. (J.Biol. Chem. (1997) 272(19): 32613-32622) havedemonstrated that three copies (3X) of the 1X enhancer element of PPE-1(containing elements ETE-C, ETE-D, and ETE-E) endows promoter sequenceswith endothelial cell specificity in-vitro, however such activity hasnot been demonstrated in-vivo.

[0089] As is well known in the art, in-vitro experiments cannot reliablypredict in-vivo results. As such, the results presented by Bu et al.,although suggestive of endothelial cell specificity, do not providesufficient evidence as to the utility of 3X enhancer element in-vivo.

[0090] The lack of in-vivo studies also brings into question theendothelial cell specificity of the 3X enhancer element in wholeorganisms. Lack of this data implies that therapeutic application ofthis element is questionable, because when employed in-vivo, and inparticular when employed for regulating angiogenesis, it is imperativethat expression of an angiogenesis regulator (e.g., cell toxin) bedirected specifically to endothelial cells, preferably in a specificsubset of endothelial cells which are involved in angiogenesis.

[0091] As is illustrated in the examples section which follows, thepresent inventors, through laborious experimentation, have provided, forthe first time, conclusive evidence as to the in-vivo activity of the 3Xenhancer element. Such evidence identifies the 3X element and itssequence derivatives (e.g., SEQ ID NO:7) as highly suitable for use intherapeutic applications.

[0092] In addition, in reducing the present invention to practice, itwas discovered that a novel configuration of the PPE-1 enhancer sequenceof the present invention endows promoter sequences with an unexpectedand highly specific activity in endothelial cells participating inangiogenesis.

[0093] Thus, according to one aspect of the present invention there isprovided an isolated polynucleotide functional as an endothelial cellspecific promoter in a mammal such as a human being.

[0094] The isolated polynucleotide includes an enhancer elementincluding one or more copies of the sequence set forth in SEQ ID NO:6and preferably one or more copies of the sequence set forth in SEQ IDNO:8, which as is illustrated in the Examples section which follows,plays an important role in regulating expression in endothelial cellsparticipating in angiogenesis.

[0095] One specific and novel sequence configuration of an enhancerelement utilizable by the present invention is illustrated in SEQ IDNO:7.

[0096] For purposes of this specification and the accompanying claims,the term “enhancer” refers to any polynucleotide sequence whichincreases the transcriptional efficiency of a promoter.

[0097] According to some embodiments of the invention, the isolatedpolynucleotide includes contiguous copies of SEQ ID NOs:6 and/or 8. Suchsequences are preferably positioned in a head-to-tail orientation,although, the enhancer element of the present invention can also includeone or more copies of a specific portion of the sequence of SEQ ID NO:6or 8, in an inverted orientation, e.g., by using sequences complementaryto SEQ ID NO:6 or 8 in construction of the enhancer element.

[0098] Preferably the isolated polynucleotide further includes anendothelial cell-specific promoter sequence element. For purposes ofthis specification and the accompanying claims, the term “promoter”refers to any polynucleotide sequence capable of mediating RNAtranscription of a downstream sequence of interest. The endothelialspecific promoter element may include, for example, at least one copy ofthe PPE-1 promoter.

[0099] Preferably, the isolated polynucleotide further includes ahypoxia response element, for example at least one copy of the sequenceset forth in SEQ ID NO: 5.

[0100] Thus, according to this aspect of the present invention, anendothelial cell specific promoter which includes various enhancerelement configurations is provided.

[0101] It will be appreciated that the enhancer element sequences can bepositioned within the promoter sequence used, upstream of the promoter,between the promoter and a downstream sequence of interest or within thesequence of interest (e.g., intron).

[0102] The isolated nucleic acid sequence of the present invention canbe used to regulate gene expression in eukaryotic tissue, and inparticular, in proliferating endothelial cells, for example endothelialcells involved in angiogenesis.

[0103] Thus, the isolated polynucleotide sequence of the presentinvention may be provided, in some cases, as part of a nucleic acidconstruct further including a nucleic acid sequence of interest which ispositioned under the regulatory control of the isolated polynucleotideof the present invention. It will be appreciated that such a nucleicacid construct can further include any additional polynucleotidesequences such as for example, sequences encoding selection markers,origin of replication in bacteria, or sequences encoding reporterpolypeptides. Such a nucleic acid construct is preferably configured formammalian cell expression and can be of viral origin. Numerous examplesof nucleic acid constructs suitable for mammalian expression are knownin the art; the Examples section which follows provides further detailof several such constructs.

[0104] For purposes of this specification and the accompanying claims,the phrase “sequence of interest” refers to any polynucleotide sequencewhich has the capacity to be transcribed by an RNA polymerase. Thisdefinition includes coding sequences translatable into polypeptides, aswell as sequence for antisense RNA, RNA which binds DNA, ribozymes andother molecular moieties which are not destined to undergo translation.Examples of nucleic acid sequence of interest which may be used by theconstruct according to the present invention are provided hereinbelowand in the Examples section which

[0105] Examples presented hereinbelow illustrate that the improvedendothelial cell specific promoters of the present invention canreliably direct expression of a reporter gene to endothelial tissueafter systemic in-vivo administration. These examples further show, forthe first time, that the isolated polynucleotide of the presentinvention can be used to preferentially express a reporter protein (GFP)in atherosclerotic and/or angiogenic tissue, thus providing for thefirst time direct evidence as to the importance of the PPE1 enhancerelement and its derivative in therapeutic applications.

[0106] While use of a reporter protein, such as GFP, may have utility indetection of early stages of metastatic tumor growth, especially inanimal models, or for non-invasive imaging of metastases (Yang, M. etal., Proc. Nat. Acad. of Sci. (2001) 27:2616-2621) such a use is only asmall portion of the projected utility of the claimed invention. It isbelieved, for example, that AdPPE-1GFP can be used in a combination withAdPPE1tk, AdPPE-1p55 and/or other anti-angiogenic treatments, in orderto follow and treat angiogenesis by a relatively non-invasive method.

[0107] Replacement of the GFP reporter gene with an apoptosis inducingfactor (e.g. p55; GenBank accession M75866) in a construct of, forexample AdPPE1-3X-p55 is predicted to reliably target apoptosis torapidly proliferating endothelial cells in angiogenic blood vessels of agrowing tumor. Because such a vector may be administered systemically,it can be employed to effectively induce apoptosis in developingmetastatic foci, without discovering the location of those foci. Such ause represents a significant improvement in comparison to prior artpractice. By inducing apoptosis specifically in developing vasculature,it is feasible to eliminate angiogenesis.

[0108] An opposite approach may be used to re-vascularize tissue, forexample in atherosclerotic patients or in patients that have sufferedsignificant impairment of peripheral circulation as a result of diseaseor injury. In this case, a construct of the type AdPPE-1-3X-GF, where GFis a growth factor (e.g., cytokine) or modificants thereof (e.g.,AdPPE-1-SEQ ID NO:7-GF), can be employed. Suitable growth factors foruse in this context include, but are not limited to, VEGF (GenBankaccession M95200) and rat PDGF— BB (GenBank accession; 99% identity tomus-AF162784) and EGR-1 (GenBank accession M22326) FGFs (including, butnot limited to, GenBank accession XM 003306) and combinations thereof.

[0109] It will be appreciated that incorporation of a hypoxia responseelement (e.g. SEQ ID NO: 5) within the promoter sequence of the presentinvention can be used to further enhance expression selectivity toIschemic tissues, thus leading to neo-vascularization of selectedtissues. As the blood supply improves, Ischemia is relieved, the hypoxiaresponse element ceases to be induced, GF levels decline and theneo-vascularization process is halted.

[0110] The promoter sequences generated according to the teachings ofthe present invention are particularly useful in regulating angiogenesisin a tissue. As illustrated in the Examples section which follows, themodified 3X (SEQ. ID. NO:7) containing promoter sequence of the presentinvention and the unmodified PPE-1 promoter are both expressed inmetastatic foci of the LLC model. However example 22 clearly illustratesthat the modified 3X sequence is specifically responsible for both adecrease in expression levels of the reporter gene in normal lung and adramatic increase in expression of the reporter gene in metastatic foci.There is neither a hint nor a suggestion in the prior art that such aresult could be achieved. Thus, use of a construct including the 3Xelement in a gene therapy context can be expected to maximize deliveryto tumors while minimizing toxic effects on surrounding normal tissue.Significantly, this is true even if the surrounding tissue contains anendothelial component, as illustrated in FIG. 37. This is because, asdemonstrated in example 11, the 3X sequence greatly increases the levelof expression in rapidly proliferating endothelial tissue, even in thecontext of the PPE-1 promoter.

[0111] For example, the p55 gene might be used in conjunction with apromoter of the present invention containing a hypoxia response elementin order to specifically induce apoptosis in growing tumors. Such astrategy is deemed feasible because a growing tumor mass tends towardischemia as tumor growth often exceeds the angiogenic capacity of thesurrounding tissue. Other expressible cell toxins which can be usedalong with the promoter sequence of the present invention in order tospecifically reduce a tumor mass include but are not limited to, otherpro-apoptotic genes, the Herpes simplex thymidine kinase gene (HSV-tk;included in the pORF—HSV1tk expression vector available from InvivoGen,San Diego, Calif.) angiostatin (Genbank accession number X05199),endostatin (Genbank accession number M33272) and angiostatin-endostatinchimera (included in the pORF—HSV1tk expression vector available fromInvivoGen, San Diego, Calif.).

[0112] Alternately, or additionally, angiostatin or endostatin genesmight be used in conjunction with a promoter of the present invention inorder to specifically block angiogenesis without inducing apoptosis.

[0113] Thus, according to alternate preferred embodiments, angiogenesismay be stimulated or blocked. This flexibility will allow varied uses ofthe invention including, but not limited to reduction of tumor mass andrevascularization of atherosclerotic regions of the heart orneo-vascularization of peripheral tissues with an inadequate bloodsupply. One relevant clinical scenario is use of a promoter according tothe present invention to generate new blood vessels to increase theblood supply in limbs of diabetic patients.

[0114] The nucleic acid construct according to the present invention canbe administered to a subject (mammals, preferably humans) per se, or ina pharmaceutical composition where it is mixed with suitable carriers orexcipients.

[0115] As used herein a “pharmaceutical composition” refers to apreparation of one or more of the active ingredients described hereinwith other chemical components such as physiologically suitable carriersand excipients. The purpose of a pharmaceutical composition is tofacilitate administration of a compound to an organism.

[0116] Herein the term “active ingredient” refers to the nucleic acidconstruct accountable for the biological effect.

[0117] Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

[0118] Herein the term “excipient” refers to an inert substance added toa pharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

[0119] Techniques for formulation and administration of drugs may befound in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition, which is incorporated herein by reference.

[0120] Pharmaceutical compositions of the present invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

[0121] Pharmaceutical compositions for use in accordance with thepresent invention thus may be formulated in conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries, which facilitate processing of the active ingredientsinto preparations which, can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

[0122] For injection, the active ingredients of the pharmaceuticalcomposition may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological salt buffer. For transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

[0123] It will be appreciated that the isolated polynucleotide of thepresent invention has been isolated based on its capacity to promote orenhance transcription in eukaryotic cells of an endothelial lineage.Therefore a mammalian cell transformed with a claimed isolatedpolynucleotide is an additional embodiment of the invention. Numerousexamples of such transformed cells are provided in examples recitedherein below.

[0124] While the examples provided hereinbelow deal specifically withthe use of the 3X sequence in conjunction with the PPE-1 promoter, it isanticipated that the enhancer sequence of the present invention willalso exert its cell specific effect when used with other eukaryoticpromoter sequences.

[0125] Such anticipation is based on prior art findings which show thatenhancer elements are often portable, i.e., they can be transferred fromone promoter sequence to another, unrelated, promoter sequence and stillmaintain activity. For examples, see D. Jones et al. (Dev. Biol. (1995)171(1):60-72); N. S. Yew et al, (Mol. Ther. (2001) 4:75-820) and L. Wu.et al. (Gene Ther. (2001) 8;1416-26). Indeed, the earlier work ofBuetal. (J.Biol. Chem. (1997) 272(19): 32613-32622) strongly suggeststhat enhancer elements related to those of the present invention, forexample enhancers including SEQ ID NO: 6 may be used with constitutivepromoters, for example the SV-40 promoter. As such, constructscontaining, methods employing and isolated polynucleotides including aeukaryotic promoter modified to include the enhancer sequence of thepresent invention are well within the scope of the claimed invention.

[0126] Thus, it is postulated that a minimal configuration of anenhancer element according to the present invention is an isolatedpolynucleotide as set forth in SEQ ID NO:8. This enhancer is anticipatedto function with a wide variety of promoters, including but not limitedto endothelial specific promoters (e.g. PPE-1; SEQ ID NO.: 1) andconstitutive promoters, for example viral promoters such as thosederived from CMV and SV-40. This enhancer should be capable of impartingendothelial specificity to a wide variety of promoters. The enhancerelement may be augmented, for example by addition of one or more copiesof the sequence set forth in SEQ ID NO:6. These additional sequences maybe added contiguously or non-contiguously to the sequence of SEQ ID NO.:8.

[0127] The present invention further includes a method of expressing anucleic acid sequence of interest in endothelial cells employing aconstruct which relies upon an enhancer element including at least onecopy of the sequence set forth in SEQ ID NO:8 and a promoter to directhigh level expression of the sequence of interest specifically toendothelial cells.

[0128] As used herein “ex-vivo administration to cells removed from abody of a subject and subsequent reintroduction of the cells into thebody of the subject” specifically includes use of stem cells asdescribed in (Lyden et al. (2001) Nature Medicine 7:1194-1201).

[0129] While adenoviruses are employed in the experiments described inexamples presented hereinbelow, the constructs of the present inventioncould be easily adapted by those of ordinary skill in the art to otherviral delivery systems.

[0130] Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

[0131] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non-limitingfashion.

[0132] Generally, the nomenclature used herein and the laboratoryprocedures utilized in the present invention include molecular,biochemical, microbiological and recombinant DNA techniques. Suchtechniques are thoroughly explained in the literature. See, for example,“Molecular Cloning: A laboratory Manual” Sambrook et al., (1989);“Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M.,ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”,John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A.Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies set forthin U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., N.Y. (1980); available immunoassays are extensivelydescribed in the patent and scientific literature, see, for example,U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

[0133] Specifically, experiments conducted in conjunction with theexamples recited hereinbelow employed the following methods andmaterials:

Materials and Methods

[0134] Cell Culture

[0135] Lewis Lung Carcinoma—(D122-96) (kindly provided by Prof. L.Eisenbach, The Weizmann Institute of Science Rehovot, Israel), HumanEmbryonic Kidney (293) and HeLa cells were grown in 4.5 gr/l DMEM,supplemented with 10% fetal calf serum (FCS), 50 U/ml penicillin, 50μg/ml streptomycine and 2 mM glutamine (Biological industries,Beit-Haemek, Israel). Bovine Aortic Endothelial Cells—BAEC (kindlyprovided by Prof. N. Savion, Goldshlager Institute, Sheba MedicalCenter, Tel-Hashomer, Israel), Normal Skin Fibroblasts—NSF, HepG2 andHuman Umbilical Endothelial Cells—HUVEC-304 (ATCC, USA) were grown in1.0gr/l DMEM (Biological industries, Beit-Haemek, Israel), supplementedwith 5% FCS, 50 U/ml penicillin, 50 μg/ml streptomycine and 2 mMglutamine. The BAEC cells were supplemented with complete fibroblastgrowth factor (Sigma, St. Louis. MO.). RINr1046-38 (RIN-38) were grownin 199 Earle's salts (5.5 mM glucose) medium supplemented with 5% FCS(Biological Industries, Beit-Haemek, Israel), 50U penicillin/ml, 50 μgstreptomycine/ml and 2 mM glutamine.

[0136] “HepG2” as used herein refers to ATCC-HB-8065.

[0137] “HeLa” as used herein refers to ATCC—CCL-2.

[0138] “Human Bronchial Epithelial cells” and “B2B” as used hereinrefers to ATCC—CRL-9609.

[0139] “HUVEC” and “Human Umbilical Vein Endothelial Cells” as usedherein refers to ATCC—CRL-1730.

[0140] “CHO” and “Chinese Hamster Ovary” as used herein refers toATCC-61.

[0141] Hypoxia Induction

[0142] Twenty six hours post transfection or transduction cells wereincubated in an isolated chamber which was washed for 30 minutes by agas flow containing 0.5% O₂, 5% CO₂, balance by N₂. The isolated chamberwas placed in humidified 5% CO₂, 37° C. incubator.

[0143] Luciferase Activity in Cells and Tissues

[0144] To assay the PPE-1 promoter activity quantitatively in-vitro andin-vivo, a Luciferase gene expression system kit was employed (PromegaCorp., Madison, Wis.). Forty eight hours post transfection ortransduction the cells were washed and 200 μl lysis buffer was added for15 minutes. Cells lysates were collected and centrifuged for 15 minutes(14,000 rpm) at 4° C. Subsequently, 10 μl of the supernatant was addedto 50 pl Luciferase assay buffer. The activity was measured inLuminometer over a 20 second period.

[0145] To assay Luciferase activity in solid tissue a 20 mg sample wasexcised and homogenized in 1 ml of the homogenization solution andcentrifuged for 15 minutes (14,000 rpm) at 4° C., and 10 ml of thesupernatant were assayed for Luciferase activity, as described above.Results were expressed as Luciferase light units per 1 μg protein.Protein was measured using the Bradford assay with bovine serum albumin(BSA) as a standard.

[0146] GFP Activity in-vitro and in-vivo

[0147] To test GFP expression in-vitro, cells were washed twice with PBSand were fixed for 30 minutes with freshly made 4% paraformaldehyde inPBS. After fixation, examination by fluorescent microscopy wasconducted.

[0148] In order to test the cellular distribution of the delivered genein-vivo, tissues were fixed in freshly made 4% paraformaldehyde in 0.1 Mphosphate buffer for 6 hours at 4° C., soaked overnight in 30% sucroseat 4° C. and frozen in OCT compound (Sakura, USA). The tissue blockswere sliced by a cryostat at 10 μm thickness and observed directly underfluorescence microscopy (FITC filter).

[0149] Proliferating and Quiescent Cells

[0150] In order to compare the PPE-1 promoter activity in proliferatingand quiescent BAEC, the cells were divided into two groups: 1.proliferating cells—growing and infecting in 10% FCS media. 2. quiescentcells—growing and infected in serum free media started in 72 hours priorto the transduction. All cells were grown in humidified incubator, 5%CO₂, 37° C.

[0151] Preparation of Recombinant Replication Deficient Adenoviruses.

[0152] Several recombinant replication deficient adenoviruses (type 5)were constructed. An expression cassette including the murinepreproendothelin-1 (PPE-1) promoter (SEQ ID NO:1) located upstream tothe Luciferase gene (originated from pGL2-basic GenBank Accession numberX65323) and the SV40 polyA site (originated from pGL2-basic GenBankAccession number X65323) was ligated into the BamHI restriction site ofpPAC.palpA (promoterless construct). The GFP gene (originated frompEGFP, GenBank accession number AAB02572) was ligated to the PPE-1promoter at the NotI restriction site. The replication deficientrecombinant adenoviruses termed Ad5PPE-1Luc or Ad5PPE-1GFP were preparedby co-transfection of pPACPPE-1Luc or Ad5PPE-1GFP with adenovirusplasmid pJM17 as described by Becker, T. C. et al. (Methods Cell biol.43, Roth M. (ed). N.Y. Academic Press, 1994, pp. 161-189) followed byharvest of recombinant virions.

[0153] Viruses were prepared for large-scale production. The viralstocks were stored at 4° C. at concentration of 10⁹-10¹² plaque-formingunits/ml (pfu/ml). The viruses Ad5CMV-Luc (kindly provided by R. Gerardfrom UTSw Dallas, Tex.) and Ad5CMV-GFP (Quantum biotechnologies,Carlsbad, Canada) containing the cytomegalovirus (CMV) immediate earlypromoter (GenBank Accession number U47119) were prepared for large scalepreparation as described for the PPE-1 viral vectors and were used as anon-tissue specific control.

[0154] Modifications of the PPE Promoter

[0155] The modified murine PPE-1 promoter was developed by insertingthree copies of the positive transcription element discovered by Bu etal (J.Biol. Chem. (1997) 272(19): 32613-32622) into the NheI restrictionenzyme site located downstream (−286 bp) to the 43 base pairs endogenouspositive element (−364 to −320 bp).

[0156] The enhancer fragment termed herein “3X” is a triplicate copy ofan endogenous sequence element (nucleotide coordinates 407-452 of SEQ IDNO:1) present in the murine PPE-1 promoter. It has been previously shownthat induction of PPE-1 promoter activity in vascular endothelial cellsdepends on the presence of this element Bu et al (J.Biol. Chem. (1997)272(19): 32613-32622). The 3X fragment was synthesized by using twocomplementary single stranded DNA strands 96 base pares in length(BioTechnology industries; Nes Tziona, Israel), (SEQ ID NO: 2 and 3).The two single stranded DNA fragment were annealed and filled usingKlenow fragment (NEB); the resulting double stranded DNA was 145 basepairs long and included Nhe-1 restriction sites (SEQ ID NO: 4).

[0157] The 3X fragment was ligated into the murine PPE-1 promoter downstream of endogenous Nhe-1 site using T4 Ligase. The resulting constructwas propagated in DH5α compatent cells and a large-scale plasmidpreparation was produced using the maxi-prep Qiagene kit.

[0158] Additional Plasmids

[0159] Wild Type PPE-1 Promoter

[0160] The PPE-1-Luciferase cassette (5249 bp) containing 1.4 kb of themurine preproendothelin-1 (PPE-1) promoter, the Luciferase gene with anSV40 polyA signal (GenBank Accession number X 65323) site and the firstintron of the murine ET-1 gene is originated from the pEL8 plasmid (8848bp) used by Harats et al (J. Clin. Inv. (1995) 95: 1335-1344). ThePPE-1-Luciferase cassette was extracted from the pEL8 plasmid by usingthe BamHI restriction enzyme, following by extraction of the DNAfragment from a 1% agarose gel using an extraction kit (Qiagen, Hilden,Germany).

[0161] The Promoter-Less pPAC.plpA Plasmid

[0162] The promoter-less pPAC.plpA plasmid (7594 bp) containingsequences of the adenovirus type 5 was originated from the pPACCMV.pLpA(8800 bp). The CMV promoter, the multiple cloning site and the SV40polyadenylation site (1206 bp) were eliminated by NotI restrictionenzyme, The fragmented DNA was extracted from 1% agarose gel. The linearplasmid (7594 bp) was filled-in by Klenow fragment and BamHI linker wasligated by rapid DNA ligation kit to both cohesive ends. The linearplasmid was re-ligated by T4 DNA ligase and transformed into DH5acompetent cells, in order to amplify the pPAC.plpA with the BamHIrestriction sites. The plasmid was prepared for large-scale preparationand purified by maxi prep DNA purification kit.

[0163] pPACPPE-1Luciferase Plasmid

[0164] The pPACPPE-1Luciferase plasmid was constructed by inserting thePPE-1-Luciferase cassette into the BamHI restriction site of thepPAC.plpA plasmid, by using T4 DNA ligase. The plasmid was subsequentlyused to transform DH5α competent cells. The plasmid (12843 bp) wasprepared for large-scale preparation and purified by maxi prep DNApurification kit.

[0165] pPACPPE-1GFP Plasmid

[0166] The pPACPPE-1 GFP plasmid was constructed by sub-cloning the GFPgene (originated from pEGFP, GenBank accession number AAB02572)downstream to the PPE-1 promoter into the NotI restriction site, by T4DNA ligase.

[0167] The plasmid was subsequently used to transform DH5α competentcells. The plasmid (11,801 bp) was prepared for large-scale preparationand purified by maxi prep DNA purification kit.

[0168] pACPPE-13X Luciferase and pACPPE-13X GFP Plasmids

[0169] The pPACPPE-1-3X Luciferase and pPACPPE-1-3XGFP were constructedby inserting the PPE-1-3XLuc or PPE-1-3XGFP cassette digested by BamHIrestriction enzyme from pEL8-3X (FIG. 26B) containing Luc or GFP intothe BamHI restriction site of the pPAC.plpA plasmid. pEL8-3X containsthe modified murine PPE-1 promoter (1.55 kb) (red)—located between BamHIand NotI that contains the triplicate endothelial specific enhancer 3X(as set forth in SEQ ID NO.: 7) located between two NheI site. Thepromoter, the Luciferase or GFP gene, the SV40 poly A sites and thefirst intron of the endothelin-1 gene, all termed the PPE-1 modifiedpromoter cassette was digested and extracted by BamHI restriction enzymeas described in materials and methods. The plasmids (12843 bp) wereprepared for large-scale preparation and purified by maxi prep DNApurification kit.

[0170] In-vitro Experiment, DNA Transduction

[0171] Cells were plated in 16 mm dishes 24 hours before transduction.DNA transduction of BAEC (Bovine Aortic Endothelial Cells), HUVEC (HumanUmbilical Vein Endothelial Cells), LLC (Lewis Lung Carcinoma) and RIN(Rat insulinoma), HepG2, HeLa and Normal skin fibroblasts (NSF) cellswas performed by incubating each cell line with 1, 5 and 10 multiplicityof infection (moi) of Ad5PPE-1Luc for 4 h in a total volume of 500 μlgrowing media, following by incubation with the growing media in a totalvolume of 2 ml for 48 hours. Ad5CMVLuc was used as a non-tissue specificcontrol.

[0172] Animals

[0173] All animal procedures were approved by the “Animal Care and UseCommittee” of Sheba Medical Center, Tel-Hashomer.

[0174] Different mouse strains were used:

[0175] (i) Male, 3 months old, wild type C57BL/6 mice (Harlan farms,Jerusalem, Israel).

[0176] (ii) Male 3 month old BALB/C mice (Harlan farms, Jerusalem,Israel).

[0177] (iii) Male and female 6 month old ApoE gene deficient micehybrids of C57BL/6x SJ129 mice (Plump AS. et al. Cell (1991)71:343-353).

[0178] (iv) Male and female 3 month old over-expressing the Luciferasegene under the control of murine PPE-1 promoter (5.9 Kb), generated byHarats et al. (J. Clin. Inv. (1995) 95: 1335-1344).

[0179] All mice were grown in the Lipids and Atherosclerosis ResearchInstitute.

[0180] Tissue Gene Expression in Normal Mice

[0181] To assay the efficiency and tissue specificity, 10¹⁰ pfu/ml ofAd5PPE1Luc or Ad5CMVLuc (as non-tissue-specific control), were suspendedin 100 μl of physiological saline and injected into the tail vein ofmice as described hereinabove. Luciferase activity was assayed 1, 5, 14,30 and 90 days post-injection. To localize cellular distribution of theexpressed reporter genes, Ad5PPE-1GFP or Ad5CMVGFP (10¹⁰ pfu/ml in 100μl physiological saline) were injected into the tail vein of normal 3month old, male C57BL/6 mice. GFP expression was detected five dayspost-injection. All mice appeared healthy and no toxicity orinflammation was noted in the liver or other tissue.

[0182] GFP Activity in Tissues

[0183] To test the cellular distribution of the delivered gene in-vivo,tissue samples from injected mice were fixed in freshly made 4%paraformaldehyde in 0.1 M phosphate buffer for 6 hours at 4° C., soakedovernight in 30% sucrose at 4° C. and frozen in OCT compound (Sakura,Calif., USA). The tissue blocks were sliced at 10 μm thickness andobserved directly under fluorescence microscopy (FITC filter).

[0184] Tumor Implantation

[0185] Lewis Lung Carcinoma cells (LLC) were harvested withtrypsin/EDTA, washed 3 times with PBS and counted with 0.1% trypan blue(Biological industries, Beit-Haemek, Israel) to assess their viability.In order to test the level of activity of the PPE-1 promoter activity intumor angiogenesis in mice, two different tumor models were used.

[0186] In the primary tumor model, the cells (1×10 ⁶ cells/ml in 100 μlphysiological saline) were subcutaneously injected to the mice backs(n=17). Twenty-one days post injection Ad5PPE-1, Ad5PPE-IGFP, Ad5CMV, orAd5CMVGFP (10¹⁰ pfu/ml) were injected into the tumor tissue (IT) orintravenously and their activity was detected as described above.

[0187] In the metastatic tumor model, the cells (5×10⁵ cells/ml in 50 μlphysiological saline) were injected to the mice foot-pad (n=12). Whenthe tumor tissue reached a size of 0.7 mm in diameter, the foot pad(with the primary tumor) was resected under anaesthetic and sterileconditions. Fourteen days post surgery the viruses (Ad5PPE-1,Ad5PPE-1GFP, Ad5CMVLuc or Ad5CMVGFP) were injected to the mouse tailvein.

[0188] In both tumor experimental models mice were sacrificed 5 dayspost viral injection, their tissues were excised and tested forLuciferase or GFP activities.

[0189] Wound Healing Model

[0190] Male 3 month old C57BL/6 mice were anaesthetized by subcutaneousinjection of sodium pentobarbital (6 mg/kg). Their backs were shaved and5 cm of straight incisions was made. The incisions were immediatelysutured by 4/0 sterile silk thread. The angiogenic process in thehealing wound was examined every two days by H&E and anti von-Willibrandantibody immunohistochemistry staining.

[0191] Ten days post incisions 10¹⁰ pfu/ml of Ad5PPE-1Luc or Ad5CMVLucwere systemically injected to the tail vein. Five days post injectionsthe mice were sacrificed and Luciferase activity was assayed asdescribed above in the skin of the incision site and in the normalcontra lateral site as a control.

[0192] Histological Examination

[0193] In order to evaluate the extent of angiogenesis in tumor andmetastasized tissue, the tissues were sliced into 5 μm sections andstained with Haematoxylin and Eosin (H & E). Anti CD31 (rat anti mouseCD31 monoclonal Ab. Pharminogen, N.J., USA) antibodies were used foranalyses of neovascularization in the tumor models.

[0194] Statistical Analysis

[0195] Analysis between groups for statistically significant differenceswas performed with the use of t-test ANOVA, or the Mann-Whitney Ranktest. Data are shown as mean+SE.

EXAMPLE 1

[0196] Analysis of 3X-PPE-1 Plasmid Activity in-vitro

[0197] In order to analyze the activity of the PPE-1-3X, a comparison ofreporter gene expression in the PPE-1-3X promoter plasmid and theunmodified PPE-1 promoter plasmid was undertaken. Reporter gene plasmidscontaining either the PPE-1-3X fragment or the unmodified PPE-1 fragmentand the reporter gene Luciferase were transfected into endothelial andnon-endothelial cell lines as well as to a bronchial epithelium cellline (B2B) which express the PPE-1 promoter (see materials and methodsabove). The B2B cell line was chosen to provide an indication of the 3Xelement's capacity to reduce expression in non-endothelial cell linesrelative to the PPE-1 promoter. Transfection was accomplished usinglipofectamine (Promega Corp., Madison, Wis.). A βgal-neo plasmid wasemployed as an indicator of the transfection efficiency in each caseaccording to accepted molecular biology practice.

[0198] Forty-eight hours post transfection, the cells were harvestedusing lysis buffer (Promega Corp., Madison, Wis.) and Luciferaseactivity was analyzed by a luminometer (TD-20e—Turner Designs,Sunnyvale, Calif.). In parallel, gal activity was analyzed in order tostandardize for different transformation efficiencies. The results aresummarized in FIG. 1 and Table 1. Luciferase activity under the controlof PPE-3X is 15-20 times higher than Luciferase activity under thecontrol of the unmodified PPE-1. In non-endothelial cell lines minimalexpression was detected using both the PPE-1 and PPE-1-3X. Thisdemonstrates that PPE-3X is a promising candidate for delivery of a genespecifically into endothelial cells in-vivo. TABLE 1 Luciferase activityin cells transfected with PPE-1 and PPE-1-3X Luciferase constructsLuciferase activity in: non endothelial endothelial cell lines celllines Plasmid HUVAC BAEC RIN PPE-1 135.12 1121.3 0.73 PPE-1-3X 76818331.7 0.32

EXAMPLE 2

[0199] Activity and Specificity of Ad5PPE-1/Luciferase in-vitro

[0200] The PPE-1/Luciferase, PPE-1-3X/Luciferase, PPE-1/GFP andPPE-1-3X/GFP were also ligated into the Ad5 plasmid to produceAd5PPE-1/Luc and Ad5PPE-1-3X/luc, Ad5PPE-1/GFP and Ad5PPE-1-3X/GFP(Varda-Bloom et al., (2001) Gene therapy 8:819-827). These constructswere assayed separately as detailed hereinbelow.

[0201] In order to test the activity of the Ad5PPE-1/luc, transfectionsof B2B (Human bronchial epithelial), BAEC (Bovine Aortic EndothelialCells) and HUVEC (Human Umbilical Vein Endothelial Cells) wereundertaken. These three cell lines express the endothelin gene and werechosen to indicate levels of expression of the tested construct in anendothelial cell. The RIN (Rat Insulinoma) cell line, which does notexpress endothelin, was employed as a negative control and transfectedwith the same construct. Ad5CMVLuc (Luciferase under the control of CMVpromoter) was used as non-endothelial-specific control in all celllines.

[0202]FIG. 2 clearly illustrates that higher Luciferase expression wasachieved in endothelial BAEC and HUVEC cell lines with the PPE-1promoter than with the CMV promoter. In the RIN cells, which are not ofendothelial origin, the CMV promoter produced more Luciferase activitythan the PPE-1 promoter. These results demonstrate the endothelialspecificity of the unmodified PPE-1 promoter.

EXAMPLE 3

[0203] Activity and Specificity of Ad5PPE-3XLuc and Ad5PPE-3XGFP

[0204] The Ad5PPE-3X/Luciferase and Ad5PPE-3X/GFP constructs were usedto transfect the cell lines described hereinabove in Example 2 in orderto ascertain the impact of the 3X element on specificity and expressionlevels. As in example 2, Ad5CMVLuc was used as anon-endothelial-specific control. Higher Luciferase expression in BAECand HUVEC cell lines was detected under the control of the PPE-3Xpromoter as compared to the CMV promoter.

[0205]FIG. 3a is a photomicrograph illustrating GFP expression under thecontrol of Ad5PPE-1-3X in the BAEC cell line. FIG. 3b is aphotomicrograph illustrating GFP expression of Ad5CMV in the BAEC line.As is clearly shown by these Figures, the PPE-1-3X promoter is moreactive in endothelial cells. These results clearly indicate that the 3Xelement does not detract from the endothelial specificity of the PPE-1promoter. Relative activities of the PPE-1 and PPE-1-3X promoters incell culture are presented in example 6 hereinbelow.

EXAMPLE 4

[0206] In-vitro Assay of Pro-Apoptotic Activity of the p55 Gene

[0207] Following sub cloning of P55 (TNFR1, GenBank accession numberM75866) into PACPPE3X (containing the PPE-1-3X promoter), and intoPACCMV, co-transfection of these plasmids and GFP (pEGFP-Cl vector;CLONTECH, Palo Alto, Calif.) was performed as described hereinabove.Briefly, the gene was subcloned downstream to the PPE-1 promoter(instead of the luciferase gene) into the NotI restriction site, by T4DNA ligase, following by transforming it into DH5α competent cells.Twenty four hours post-transfection, small and rounded apoptotic cellswere visually discernible from normal cells. Electron microscopy ofcells transfected with the pro-apoptotic plasmids showed typicalappearance of apoptosis, confirming the visual evaluation.

[0208] Under the control of the PPE-1-3X promoter, apoptosis was inducedby p55 only in endothelial cells (FIG. 4), whereas the CMV promoter didnot show any cell specific activity. Luciferase under the control ofPPE-1-3X did not induce apoptosis in any tested cell lines. Theseresults indicate that by employing the PPE-1-3X promoter, it is feasibleto induce apoptosis specifically in endothelial cells.

EXAMPLE 5

[0209] Hypoxia Responsive Element (HRE) can Enhance Target GeneExpression in Hypoxic Sensitive Endothelial Cells

[0210] Hypoxia is an important regulator of blood vessels' tone andstructure. It has also been shown to be a potent stimulus ofangiogenesis (in both ischemic heart diseases and cancer (Semenza, G. L.et al. (2000) Adv Exp Med Biol.; 475:123-30; Williams, K. J. (2001)Breast Cancer Res. 2001: 3;328-31 and Shimo, T. (2001) Cancer Lett.174;57-64). Further, hypoxia has been reported to regulate theexpression of many genes including erythropoietin, VEGF, glycolyticenzymes and ET-1. These genes are controlled by a common oxygen-sensingpathway, an inducible transcription complex termed hypoxia induciblefactor-1 (HIF-1). The HIF-1 complex mediates transcriptional responsesto hypoxia by binding the cis acting hypoxia responsive element (HRE) oftarget genes. The HRE is a conserved sequence located in the promotersof few genes that respond to hypoxia including: VEGF, Nitric OxideSyntase-2, erytropoietin and others including endothelin-1, ET-1. TheET-1 promoter contains an inverted hypoxia response element atposition—118 bp upstream of the transcription start site, the elementcontain 7 base pairs and is located between the GATA-2 and API sites 5′GCACGTT 3′—50 base-pairs. (SEQ ID NO: 5.)

[0211] The preproendothelin-1 (PPE-1) promoter contains an hypoxiaresponsive element (HRE) that has the potential to increase itsexpression in the hypoxic microenviroment of tumor or ischemic tissues,thus making it “tumoral tissue specific” and/or “ischemic tissuespecific”. In order evaluate the actual function of this HRE, assays ofthe PPE-1 promoter and PPE-1-3X promoter in conjunction with aLuciferase or GFP reporter gene and delivered by an adenoviral vectorwere undertaken.

[0212] Luciferase activity under the control of the PPE-1 promoter orthe PPE-1-3X promoter was compared in BAEC cells under normoxic andhypoxic conditions (0.5% O₂ for 16 h). The Luciferase activity under thecontrol of PPE-1 promoter was 5 times higher when exposed to hypoxia(FIGS. 5 and 6). Further, the Luciferase activity under the control ofPPE-1-3X promoter was 2.5 times higher under hypoxic conditions. Insummary, introduction of the 3X element into the PPE 1 promoter is tillcapable of increasing expression levels of a downstream gene in responseto hypoxia, even though the normoxic levels of expression with thePPE-1-3X gene are higher than those observed with the unmodified PPE-1promoter.

EXAMPLE 6

[0213] Further Evaluation of PPE-1-3X and PPE-1 Promoter Activity inEndothelial Cell Lines

[0214]FIG. 7 summarizes the results from B2B, HUVEC and BAECtransfection experiments using pPPE-1/Luciferase andpPPE-1-3X/Luciferase. Higher Luciferase expression (30, 8.5 and 1.5times more) was observed under the control of the PPE-1-3X promoter thanunder the PPE-1 promoter in B2B, HUVEC and BAEC, respectively. Theseresults confirm those presented hereinabove and serve to establish thatPPE-1-3X is well suited to directing high level expression specificallyto endothelial cells. In the context of future in-vivo delivery, thehigher levels of expression achieved with the PPE-1-3X constructtranslate into administration of smaller amounts of DNA. This, in turn,will serve to increase specificity even further.

EXAMPLE 7

[0215] Efficiency, Specificity and Stability of Ad5PPE-1Luc in-vivo

[0216] In order to confirm that the endothelial specificity ofexpression observed in examples 2 through 6 was not an artifact of cellculture, the Ad5PPE-1/Luciferase construct was injected into C57BL/6mice as described hereinabove in “Tissue gene expression in normalmice”. As in the in-vitro studies, Ad5CMV/Luciferase was employed as anegative control.

[0217] Following injection of adenoviral vectors, the specific activityand stability of Luciferase in vascularized and non-vascularized tissueswas assayed. Results are summarized in FIG. 8 (Luciferase expressionrelative to expression in liver) and Table 2 (Luciferase expression as apercentage of total expression in the body). As expected, inAd5CMV/Luciferase treated mice most of the Luciferase activity (>80% ofthe total body expression) was found in the liver. Luciferase activitycontrolled by the PPE-1 promoter was lower in the liver (37-54% of thetotal body expression). The PPE-1 derived expression was much higher inthe aorta (23-33% of the total body expression 5 and 14 days postinjection, respectively), compared to Ad5CMV/Luciferase. treated mice(up to 1.8% of total body expression; Table 2). These results confirmthe endothelial specificity observed in cell culture. It should beremembered that the liver is a highly vascularized organ. Thereforeexamination of cellular expression within organs was undertaken, asdetailed hereinbelow. TABLE 2 Luciferase expression in organs 5 and 14days post injection of PPE-1 and CMV based constructs Day post injection5 14 Light units/μg protein Light units/μg protein Organ PPE-1 CMV PPE-1CMV Aorta 13.0 ± 2.9  1.4 ± 0.5 10.6 ± 2.4  1.3 ± 0.3 (32.7%) (0.56%)(12.6%) (1.1%)  Heart 0.2 ± 0.1   1 ± 0.6 1.5 ± 0.3 1.8 ± 0.6 (0.5%) (0.4%)  (1.7%)  (1.6%)  liver 22.7 ± 4.5    219 ± 111.5 34.9 ± 7.8  52.8± 10.6 (57%)  (88.6%) (41.6%) (46.8%) lung 0.2 ± 0.1 2.3 ± 1.0 3.6 ± 0.82.0 ± 0.9 (0.5%)  (0.9%)  (4.3%)  (1.8%)  muscle 0.3 ± 0.1 0.8 ± 0.2 1.2± 0.3 1.5 ± 0.5 (0.7%)  (0.3%)  (1.4%)  (1.3%)  spleen 1.3 ± 0.8 1.6 ±0.9 2.0 ± 0.4 2.3 ± 0.9 (3.2%)  (0.6%)  (2.4%)  (2.0%)  pancreas   2 ±0.6 20.1 ± 6.8  26.4 ± 5.9  45.2 ± 24.5 (5.0%)  (8.1%)  (31.5%) (40.1%)kidney 0.1 ± 0   0.9 ± 0.6 0.6 ± 0.1 0.8 ± 0.3 (0.25%) (0.4%)  (0.71%) (0.7%) 

[0218]FIGS. 30A and 30B demonstrate the absolute Luciferase activity(light units/μg protein) in the aortas (A) and livers (B) of the 110injected mice. Luciferase activity was measured 1 (n=13), 5 (n=34), 14(n=32), 30 (n=20) and 90 (n=11) days post injection. The results in theaorta represent the promoters (PPE-1 or CMV) activity mostly inendothelial cells, while the results in the livers represent theiractivity mostly in hepatocytes.

EXAMPLE 8

[0219] Assays of Efficiency, Specificity and Stability of Ad5PPE-1in-vivo—in BALB/C Mice

[0220] The experiments of example 7 were repeated in 12 week old BALB/Cmice (n=10 for each group) in order to demonstrate that the observedresults were not an artifact of a particular strain of animals.

[0221] Because Absolute results with the adenoviral vectors were lowerin BALB/C mice than in C57BL/6 mice, the Luciferase expression isexpressed as percentage of the total Luciferase activity in all tissues.

[0222] The highest relative Luciferase expression 5 days post injectionwas observed in the spleens of Ad5PPE-1 (90.9%), and in the livers ofAd5CMV (86.2%) injected mice. A significant increase in the relativeLuciferase activity in the aortas of Ad5PPE-1 injected mice 14 days postinjection (32.9%), compared to its activity five days post injection(1.75%) was also observed (FIGS. 31A and 31B; Ad5PPE-1Luc—open bars;Ad5CMVLuc-black bars).

[0223] These results confirm that regardless of mouse strain, the tissuespecificity of the PPE-1 promoter is sufficiently strong to effectivelyeliminate hepatocyte expression, despite preferential uptake of injectedDNA by hepatocytes.

EXAMPLE 9

[0224] Cellular Localization of Gene Delivered by Ad5PPE-1 in-vivo

[0225] In order to ascertain cellular expression sites of the geneexpressed by PPE-1 in-vivo, Green Fluorescent Protein (GFP) delivered bythe adenoviral vector Ad5PPE-1-GFP was used. Ad5CMVGFP (Quantum, Canada)was used as non-endothelial-cell-specific negative control. Five dayspost-intravenous injection the mice were sacrificed and their tissueswere analyzed by fluorescent microscopy.

[0226] In the mice injected with Ad5CMVGFP vector, most of theexpression was detected in the hepatocytes, and no expression wasdetected in endothelial cell in the liver (FIG. 9A). In sharp contrast,Ad5PPE-1-GFP injected mice (FIG. 9B), showed no expression inhepatocytes, but significant expression in endothelial cells in theblood vessels of the liver. Similar results were obtained in othertissues where practically all the PPE-1 derived expression was detectedin the endothelium, while none of the CMV derived expression wasendothelial. These results indicate endothelial specificity is preservedeven within an organ containing endothelial and non-endothelial cells.This finding has important implications for prevention of angiogenesisin growing tumors.

EXAMPLE 10

[0227] Assays of Efficiency and Endothelial Specificity ofAd5PPE-1-3XLuc and Ad5PPE-1-3X GFP in-vitro

[0228] In order to determine the relative efficacy of Ad5PPE-1 andAd5PPE-1-3X in driving expression of the reporter genes Luciferase andgreen fluorescent protein (GFP) in cells, specific activity inendothelial cells was tested in-vitro using cell lines describedhereinabove. Ad5CMVLuc and Ad5CMVGFP were employed as non-tissuespecific controls. Ad5PPE-1Luc and Ad5PPE-IGFP were employed toascertain the relative change in expression level caused by addition ofthe 3X sequence.

[0229] Results, summarized in FIGS. 10 and 11, indicate that Luciferaseactivities under the control of the PPE-1-3X promoter were 5-10 timeshigher in EC lines (Bovine Aortic Endothelial Cells—BAEC) compared toactivity in non-endothelial cells—Rat Insulinoma—RIN, HeLA, HePG2 andnormal skin fibroblasts (NSF) (FIGS. 10 and 11).

[0230]FIG. 10 shows Luciferase activity as light units/μg protein inB2B, BAEC and RIN cells transduced by Ad5PPE-1Luc, Ad5PPE-1-3XLuc, andAd5CMVLuc Highest Luciferase expression was observed in RIN cellstransduced by Ad5CMVLuc, however this construct was poorly expressed inBAEC and B2B cells. The next highest level of Luciferase expression wasobserved in BAEC cells transduced by Ad5PPE-1-3XLuc. Ad5PPE-1Luc wasexpressed at lower levels in BAEC cells. In the B2B cell lineAd5PPE-1Luc and Ad5PPE-1-3XLuc were expressed at nearly identicallevels.

[0231] Overall, Luciferase activity in the endothelial cell lines underthe control of PPE-1-3X promoter was 23 times higher than under thecontrol of PPE-1 promoter and 23-47 times higher than under the controlof the CMV promoter at the same infection conditions (moi=10). This isdespite the fact that Luciferase expression in non-endothelial RIN cellswas 3000 times higher under the control of the CMV promoter (FIG. 10).

[0232] In order to establish that PPE-1 and PPE-1-3X are inactive inother non-endothelial cell lineages HeLA, HepG2, NSF cell lines weretransduced. BAEC was employed as an endothelial control. FIG. 11 showsLuciferase activity as light units/μg protein in HeLA, HepG2, NSF andBAEC cells transduced by Ad5PPE-1Luc, Ad5PPE-1-3XLuc and Ad5CMVLuc.Transduction with Ad5CMVLuc caused high levels of Luciferase expressionin HeLA, HepG2 and NSF cells. These cell lines failed to expressLuciferase under the control of PPE-1 and expressed Luciferase at lowlevels with the PPE-1-3X promoter. As expected, BAEC cells transducedwith Ad5PPE-1Luc or Ad5PPE-1-3XLuc exhibited high Luciferase expression.

[0233] Taken together these results indicate that introduction of the 3Xsequence into the PPE-1 promoter caused higher levels of expression inendothelial cell lines while preventing unwanted expression innon-endothelial cells.

[0234] Addition of the 3X sequence to the PPE-1 promoter also increasedlevels of Green fluorescent protein expression in EC lines (BovineAortic Endothelial Cells—BAEC) as indicated in FIGS. 12A-C which depictsGFP expression in BAEC transduced by moi=1. No expression of GFP wasobserved using a CMV promoter in this experiment.

[0235] In FIG. 12, panel A indicates Ad5PPE-1-3XGFP transduced cells,panel B indicates Ad5PPE-1GFP transduced cells and panel C indicatesAd5CMVGFP. Again, introduction of the 3X sequence into the PPE-1promoter significantly increased expression of the reporter gene. Thisresult indicates that the ability of the 3X sequence to function as anendothelial specific enhancer is not a function of the downstream genebeing transcribed.

[0236] Moreover, Ad5PPE-1-3X-GFP and Ad5PPE-1GFP transduction resultedin no GFP expression in non-endothelial cells SMC, HelA, HePG2 andnormal skin fibroblasts (NSF) compared to the high expression under theCMV promoter as summarized in FIGS. 13-16.

[0237]FIG. 13 shows GFP expression in SMC transduced by moi=1 of eitherAd5PPE-1-3XGFP (panel A) or Ad5CMVGFP (panel B). While high level GFPexpression resulted from Ad5CMVGFP transduction, no GFP expressionresulted from transduction with Ad5PPE-1-3XGFP transduction.

[0238]FIG. 14 shows results of a similar experiment conducted in HeLacells. As in the previous figure, panel A indicates cells transducedwith Ad5PPE-1-3XGFP and panel B indicates cells transduced withAd5CMVGFP. Again, while high level GFP expression resulted fromAd5CMVGFP transduction, no GFP expression resulted from transductionwith Ad5PPE-1-3XGFP transduction.

[0239]FIG. 15 shows results of a similar experiment conducted in HepG2cells. As in the previous figure, panel A indicates cells transducedwith Ad5PPE-1(3X)GFP and panel B indicates cells transduced withAd5CMVGFP. Again, while high level GFP expression resulted fromAd5CMVGFP transduction, no GFP expression resulted from transductionwith Ad5PPE-1-3XGFP.

[0240]FIG. 16 shows results of a similar experiment conducted in NSFcells. As in the previous figure, panel A indicates cells transducedwith Ad5PPE-1-3XGFP and panel B indicates cells transduced withAd5CMVGFP. Again, while high level GFP expression resulted fromAd5CMVGFP transduction, very low GFP expression resulted fromtransduction with Ad5PPE-1-3XGFP.

[0241] These results, taken together, indicate a high level ofendothelial specificity and a high level of endothelial expression isobtained by using a modified PPE-1 promoter containing the 3X sequenceof SEQ ID NO.: 7.

EXAMPLE 11

[0242] Cellular Localization of a Reporter Gene Delivered by Ad5PPE-1-3Xin-vivo

[0243] In order to determine the cellular localization pattern of areporter gene expressed under the control of the PPE-1-3X promoterin-vivo, Ad5PPE-13XGFP and Ad5PPE-1GFP were injected into mice asdescribed hereinabove. Five days post-intravenous injection, the micewere sacrificed and their tissues were analyzed by a fluorescentmicroscopy.

[0244] Significantly higher GFP activity was observed in the endothelialcells of the liver, kidney and spleen blood vessels of Ad5PPE-1-3XGFPinjected mice compared to the Ad5PPE-1GFP injected mice. FIGS. 17A and Bshow representative results.

[0245]FIG. 17A shows low level GFP expression in endothelial cellslining a blood vessel of a mouse injected with the Ad5PPE-1GFP. FIG. 17Bshows the much higher level of GFP expression resulting from addition ofthe 3X sequence to the construct.

[0246] Despite the high expression in the lining of the blood vessels,no expression was detected in the hepatocytes, glomeruli, epithelialcells and splenocytes (FIGS. 18 and 19).

[0247]FIG. 18 shows representative results from kidney tissue ofinjected mice. Ad5CMVGFP injected mice (FIG. 18A), Ad5PPE-1GFP (FIG.18b) and Ad5PPE-1-3XGFP (FIG. 18C) injected mice all exhibited low GFPactivity in kidney cells. In FIG. 18B, slightly higher GFP expression isvisible in the blood vessel wall (indicated by arrow).

[0248]FIG. 19 shows representative results from spleen tissue ofinjected mice. Ad5CMVGFP injected mice (FIG. 19A), Ad5PPE-1GFP injectedmice (FIG. 19B) and Ad5PPE-1-3XGFP injected mice (FIG. 19C) allexhibited low level GFP activity in cells of the spleen. Higher GFPactivity is visible in the blood vessels of Ad5PPE-1-3XGFP injected mice(indicated by arrow).

[0249] These results confirmed that both the PPE-1 and the PPE-1-3Xpromoter are endothelial cell specific in-vivo. They further suggestthat activity of both promoters was limited in non-proliferatingendothelial tissue (i.e. blood vessels of healthy organs. Therefore,assays in a tumor angiogenic model were undertaken.

EXAMPLE 12

[0250] Assays of the Ad5PPE-1 Construct in Tumor Neovascularizationin-vivo

[0251] In order to ascertain the ability of AD5PPE to specificallydirect expression of a reporter gene to angiogenic blood vessels in atumor, the murine LLC model (described hereinabove in materials andmethods) was employed.

[0252] In a one experiment, Luciferase expression in tumorneovascularization was tested five days post systemic injections ofAd5PPE-1Luc or Ad5CMVLuc (10¹⁰ pfu/ml each).

[0253] In this experiment, systemic injection of Ad5CMVLuc to bothprimary and metastatic tumor models resulted in minimal expression inthe primary tumor or in the metastatic lung. This level of expressionwas similar to the minimal expression of Luciferase directed by CMV innaive normal lungs (FIG. 35; black bars; n-12). In sharp contrast, underthe control of PPE-1 promoter (FIG. 35; open bars; n=9), the highlyangiogenic lung metastases were associated Luciferase activity which wasabout 200 times higher than the Luciferase activity in thepoorly-vascularized primary tumor and the naive lungs.

[0254] The Luciferase expression in non-metastatic tissues such as theliver, kidney, heart and pancreas was minimal. The expression level inthe aorta was about 30% of the levels in the metastatic lungs.

[0255] In an additional experiment in the LLC model Ad5PPE-1GFP andAd5CMVGFP constructs were employed to localize reporter gene expressionin the primary tumor and metastatic lungs.

[0256] Ad5PPE-1GFP injected mice, showed high levels of GFP specificexpression in the blood vessels of the primary tumor (FIG. 36C),although no expression was detected in the tumor cells themselves. Thisobservation is consistent with the results of the LLC cell culture modelpresented in example 20. In lung metastases, high levels of GFPexpression were detected in both big arteries and small angiogenicvessels of the metastatic foci (FIG. 36A). No expression was detected inthe normal lung tissue. The endothelial cell localization wasdemonstrated by co-localization of the GFP expression (FIG. 16A) and theCD31 antibody immuno-staining (FIG. 16B). In striking contrast, inAd5CMVGFP injected mice, no GFP activity was detectable in both theprimary tumor and lung metastasis.

[0257]FIG. 36C illustrates GFP expression in blood vessels of a primarytumor after intra tumoral injection of Ad5PPE-1GFP. FIG. 36D is a phasecontrast image of the same filed as panel C illustrating the tumor andits blood vessels.

[0258] These results indicate that while PPE-1 does not drive high levelexpression in tumor cells per se, the promoter does drive high levelexpression in vascular endothelia within the tumor, especially inrapidly proliferating angiogenic vessels.

[0259] Intra-tumor injection of Ad5CMV into primary subcutaneous tumormodel resulted in high Luciferase expression in the tumor tissue andmoderately levels of expression liver (10% of the amount expressed inthe tumor; FIG. 42). No expression was detected in the metastatic lungs.On the other hand, when injected intra-tumoral, Luciferase expressionunder the control PPE-1 promoter resulted in similar Luciferase levelsof expression in the primary tumor and the metastatic lungs and noexpression was detected in the liver.

EXAMPLE 13

[0260] Assays of the Ad5PPE-1 Construct in a Carcinoma Cell CultureSystem

[0261] In order to assay the efficiency of Ad5PPE-1 and Ad5CMV to driveLuciferase expression in cancerous cells, the D122-96 Lewis LungCarcinoma cell line was employed.

[0262] In-vitro transduction at varying multiplicities of infection(moi) was performed. The results indicate that both adenoviral vectorsare able to transduce the Luciferase gene to these cells (Table 3).Nevertheless, Luciferase activity directed by the PPE-1 promoter wasmuch lower in the LLC cells than the activity detected in endothelialcells, 50 vs. 1000-2500 light units/μg protein, respectively. TABLE 3In-vitro transduction of Lewis lung carcinoma cell line (D122-96) withAd5PPE-1Luc and Ad5CMVLuc. MOI = 1 MOI = 5 MOI = 10 Ad5PPE-1 8.1 ± 0.0633.95 ± 7.0 50.7 ± 5.0 Ad5CMV 9.3 ± 1.1   47.3 ± 4.0 88.13 ± 10.1

EXAMPLE 14

[0263] Assay of the Effect of the 3X Sequence in Tumor Angiogenic BloodVessels in-vivo

[0264] In order to ascertain the effect of the 3X sequence on the PPE-1promoter in angiogenic blood vessels, the Lewis Lung Carcinoma (LLC)metastases model (described hereinabove in material and methods) wasemployed. Five days post IV injection of 10¹⁰ infectious units ofAd5PPE-1GFP, Ad5PPE-1-3XGFP or Ad5CMVGFP, the mice were sacrificed andtheir tissues were analyzed as described in material and methods.

[0265] FIGS. 20A-D summarize the GFP expression in metastatic lungs ofcontrol mice injected with Saline (FIG. 20A), mice injected withAd5CMVGFP (FIG. 20B), mice injected with Ad5PPE-1GFP (FIG. 20C) and miceinjected with Ad5PPE-1-3XGFP (FIG. 20D). Anti-CD31 immunostaining (FIGS.20C′ to 20D′) confirm the location of the GFP expression in eachmetastatic tissue. The results show that while no GFP expression wasdetected in control—saline injected mice (FIG. 20A), there was a slightexpression around the epithelial bronchi of the CMV injected mice, butnot in the angiogenic blood vessels of the metastatic lung of these mice(FIG. 20B). Low GFP expression was observed in metastatic lungs ofAd5PPE-1GFP injected mice (FIGS. 20C and 20C′), while high and specificexpression was observed in the new blood vessels of Ad5PPE-1-3XGFPinjected mice (FIGS. 20D and 20D′).

[0266] These results explain the apparent disparity between the in-vivoresults of example 10 and the in-vitro results of examples 2, 3 and 6.Both the PPE-1 and the PPE-1-3X promoter are endothelial specific.However, the 3X sequence greatly increases the level of expression inrapidly proliferating endothelial tissue, such as newly forming bloodvessels in a growing tumor.

EXAMPLE 15

[0267] Effect of the 3X Element on the PPE-1 Promoter in TumorAngiogenic Blood Vessels

[0268] In order to study the effect of the 3X element of the presentinvention on efficacy and specific activity of the PPE-1 promoter intumor angiogenic blood vessels, the LLC metastases model was employed.Five days post i.v. injection of 10¹⁰ pfu/ml of Ad5PPE-1Luc,Ad5PPE-1-3XLuc, Ad5CMVLuc, Ad5PPE-1GFP, Ad5PPE-1-3X-GFP or Ad5CMVGFP,the mice were sacrificed and their tissues were analyzed for Luciferaseor GFP expression as described hereinabove.

[0269]FIG. 37 is a histogram comparing Luciferase expression in normallungs versus that in metastatic lungs after systemic injection ofAd5PPE-1-3Xluc, Ad5PPE-1Luc or Ad5CMVLuc. Experimental groups wereAd5CMVLuc (n=7; black bars), Ad5PPE-1Luc (n=6; gray bars) andAd5PPE1-3XLuc (n=13; brown bars). Activity is expressed as lightunits/μg protein.

[0270] Luciferase expression under the control of the PPE-1-3X promoterwas 35 fold greater in the metastatic lungs relative to its activity innormal lungs and 3.5 fold higher than expression driven by the PPE-1promoter without the 3X element (p<0.001). Very low Luciferase activitywas detected in other tissues of mice injected with Ad5PPE-1-3XLuc.Calculating the Luciferase expression in the lungs as percentage fromthe liver of each injected animal revealed that the activity increased10 fold in the metastatic lung compared to the activity in normal lung(FIG. 38).

[0271] In order to localize reporter gene expression to specific celltypes, GFP constructs were employed. FIG. 39 shows the GFP expression(FIG. 39A) in metastatic lungs of Ad5PPE-1-3XGFP injected mice.Immuno-staining by CD31 antibody (FIG. 39B) confirm the location of theGFP expression in the new blood vessels. No GFP expression was detectedin control—saline injected mice. Low level expression around theepithelial bronchi of the CMV injected mice, but not in the angiogenicblood vessels of the metastatic lung. In summary, these results indicatethat large increases in expression level resulted from introduction of a3X element into Ad5PPE-1 constructs and that this increased expressionwas specific to the angiogenic blood vessels of tumors. Potentially, theobserved effect may be coupled with the hypoxia response describedhereinabove to further boost expression levels of a sequence ofinterest.

EXAMPLE 16

[0272] Further Characterization of the PPE-1 Hypoxia Response

[0273] In order to further characterize the effect of hypoxia on themurine PPE-1 promoter activity, bovine aortic endothelial cells (BAEC)were transfected by a DNA plasmid (pEL8; FIG. 26A). The pEL8 plasmidcontains the murine PPE-1 promoter (1.4 kb) (red), the luciferase gene(1842 bp), the SV40 poly A sites and the first intron of theendothelin-I gene, all termed the PPE-1 promoter cassette was digestedand extracted by BamHI restriction enzyme as described in material andmethods. After transfection, cells were subjected to hypoxic conditions.

[0274] Luciferase expression in transfected BAEC subjected to 18 hoursof hypoxia (0.5% O2) was eight times higher than Luciferase expressionin cells grown in a normoxic environment (FIG. 21). FIG. 21 shows thatLuciferase activity (light units/μg protein) in BAEC transfected by aplasmid containing the murine PPE-1 promoter was significantly higherwhen transfected cells were incubated in a hypoxic environment.Equivalent transfection efficiencies were confirmed by co-transfectionwith a α-galactosidase reporter vector and assays of LacZ activity.

[0275] In order to determine whether murine PPE-1 promoter delivered byadenoviral vector is also up-regulated by hypoxia, BAEC were transducedby Ad5PPE-1Luc. Ad5CMVLuc was used a non specific control in thisexperiment. Results are summarized in FIG. 22. Hypoxia Luciferaseactivity in BAEC transduced by Ad5PPE-1Luc. In stark contrast, nosignificant difference between normoxia and hypoxia was detected in theAd5CMV transduced cells (FIG. 22).

[0276] To understand whether the enhancement of the PPE-1 promoteractivity is specific to endothelial cells, different cell lines (BAEC,B2B, CHO, RIN and Cardiac Myocytes) were transduced by Ad5PPE-1 (moi=10)and were subjected to hypoxia (0.5% O₂) or normoxia environment. Resultsare summarized in FIG. 23. Luciferase expression was slightly increasedin B2B cells and significantly increased in BAEC cells cultured in ahypoxic environment. Luciferase expression in other cell lines wasreduced by the hypoxic environment, compared to normoxia. These resultsconfirm that hypoxic induction of the PPE-1 promoter occurs primarily inendothelial cell lineages.

EXAMPLE 17

[0277] Effect of the 3X Sequence on the PPE-1 Hypoxia Response

[0278] In order to ascertain the effect of the 3X sequence on the PPE-1hypoxia response, BAEC were transduced by Ad5PPE-1Luc andAd5PPE-1(3X)Luc. Following transduction, the BAEC cells were incubatedeither in a hypoxic or a normoxic environment as detailed hereinabove.Results are summarized in FIG. 24. Luciferase expression using theAd5PPE-1Luc construct significantly increased (seven folds) in responseto hypoxia (2578 in hypoxia and 322.1 in normoxia). In contrast, theAd5PPE-1(3X)Luc construct exhibited only 1.5 fold increase in responseto hypoxia (from 2874.5 in normoxia to 4315 in hypoxia conditions).These results indicate that the high normoxic level of expressionobserved when the 3X sequence is added to the PPE-1 promoter serves tomask the hypoxic response to some extent.

EXAMPLE 18

[0279] Assays of the PPE-1 Response to Hypoxia in a Transgenic MouseModel

[0280] In order to examine the murine PPE-1 promoter activity in tissuessubjected to regional hypoxia/ischemia, mPPE-1-Luc transgenic mice,described hereinabove in materials and methods, were employed. The micewere induced to regional hind limb ischemia as previously described(Couffinhal T. et al. (1998) Am. J. Pathol. 152;1667-1679). In brief,animals were anesthetized with pentobarbital sodium (40 mg/kg, IP).Unilateral ischemia of the hind limb was induced by ligation of theright femoral artery, approx. 2 mm proximal to the bifurcation of thesaphenous and popliteal arteries. To verify the induction of functionalchange in perfusion, ultrasonic imaging was performed on days 4 and 14by Synergy ultrasound system (GE) equipped with a 7.5 MHz transducer andangiographic software. Animals were housed under conventional conditionsfor up to 18 days.

[0281] Luciferase expression was assayed 2, 5, 10 and 18 days postligation in the ischemic muscle, in the normal non-ligated muscle, inthe liver, lung, and aorta.

[0282] Results, summarized in FIG. 25, show that while no significantdifference was detected in the liver, lung and aorta during the dayspost ligation, Luciferase gene expression increased after the femoralligation in both in the normal non-ligated and in the ischemic muscle.While peak Luciferase expression in the ischemic muscle was detectedfive days post ligation, peak Luciferase expression in the non-ligatedmuscle was detected ten days post femoral artery ligation. Thisindicates that the hypoxic response of the PPE-1 promoter is functionalin an in-vivo system. Luciferase expression in the non-ischemic muscledid not change during the days tested, compared to its expression in thecontrol non-operated tissue (day=0). In contrast, Luciferase expressionin the ischemic muscle was significantly higher on day 5 than at othertime points.

[0283] On day 5, PPE-1 driven expression of Luciferase was 2.5 timeshigher than in control non-operated mice and compared to the ischemicmuscle in days 10 and 18 (FIG. 40).

[0284] Expression of Luciferase in other non-ischemic tissues includingliver, lungs and aorta of the transgenic mice subjected to regionalischemia revealed no significant changes within 18 days post ischemicinduction in the Luciferase expression in these tissues (FIG. 41).

[0285] Further, these results confirm that Luciferase expression washigher in tissues containing a high percentage of endothelial tissue(lung and aorta) than in those tissues containing a low percentage ofendothelial tissue (liver and non-ischemic muscle).

EXAMPLE 19

[0286] Effect of Level of Cellular Proliferation on Ad5PPE-1Luc Activityin Endothelial Cells

[0287] In order to ascertain the effect of level of cellularproliferation on efficiency and specific activity of Ad5PPE-1Luc, anangiogenic model of endothelial cells (BAEC), was tested in-vitro.Transduced BAEC were either induced to quiescence by serum deprivationor grown in 10% FCS for normal proliferation. Briefly, cells weretransduced for 48 hours either as quiescent cells—72 hours post serumdeprivation or as proliferating cells—in normal media (10% FCS).Luciferase activity is expressed as light unit/μg protein, to normalizefor the difference in cell amount. The results presented are an averageof triplicate test from four representative independent experiments.

[0288] Luciferase expression under the control of PPE-1 promoter (openbars; FIG. 28) was 4 times higher in normal proliferating BAEC than inquiescent cells, and 25 times higher in normal proliferating BAEC thanLuciferase expression under control of the CMV promoter (Black bars;FIG. 28). Further, in proliferating cells, the activity under thecontrol of PPE-1 promoter was 10 times higher than that under the CMVpromoter control.

[0289] In order to simulate angiogenic conditions in-vitro, Ad5PPE-1Lucactivity was tested in BAEC induced to rapid proliferation by additionof 40 ng/ml vascular endothelial growth factor (VEGF). Activity underthese conditions was compared activity in normal proliferating cells andquiescent cells as described hereinabove. Luciferase expression in BAECinduced to cell proliferation with VEGF was 44 times higher than innormal proliferating cells, and 83 times higher than in quiescent cells(FIG. 29).

[0290] Together, these experiments indicate that the level of activityof a sequence of interest under transcriptional control of the PPE-1Promoter is a function of the level of cellular proliferation, withrapid proliferation causing higher levels of expression.

EXAMPLE 20

[0291] Assays of the PPE-1 Promoter in Atherosclerosis Induced Mice

[0292] In order to test the efficiency and specificity of the Ad5PPE-1vector in atherosclerotic blood vessels, 10¹⁰ pfu/ml of the viralvectors were systemically injected to 6 month old ApoE deficient mice(Plump, A. S. et al. Cell; 1991; 71 :343-353).

[0293] As ApoE deficient mice age, they develop high cholesterol valuesand extensive atherogenic plaques with no induction of lipid reach diet.FIG. 32 is a picture of an aorta dissected from an ApoE deficient mousecolored by Sudan-IV. Note that the thoracic aorta contains less redstained atherosclerotic lesions while the abdominal region is highlyatherosclerotic. (FIG. 32 adapted from Imaging of Aortic atheroscleroticlesions by ¹²⁵I-HDL and ¹²⁵I-BSA. A. Shaish et al,Pathobiology—submitted for publication).

[0294]FIG. 33 summarizes Luciferase expression observed 5 days postsystemic injections of Ad5PPE-1Luc (open bars; n=12) and Ad5CMVLuc(black bars; n=12) to ApoE deficient mice. Results are presented asabsolute Luciferase expression in the thoracic area that contains lessatherosclerotic lesion, and the abdominal aorta that is richatherosclerotic lesion.

[0295] Luciferase expression controlled by the PPE-1 promoter was 6 foldhigher in the highly atherosclerotic abdominal, and 1.6 fold higher inthe slightly atherosclerotic thoracic aorta as compared to expressionunder the control CMV promoter.

[0296] No significant difference was observed between the two aortaregions in the Ad5PPE-1Luc injected mice, while higher Luciferaseexpression was observed in thoracic aorta of the Ad5CMVLuc injectedgroup compared to low expression in the abdominal aorta that containlesion.

[0297] These results indicate that while a constitutive promoter (CMV)has a tendency to shut down in areas where atherosclerosis is mostsevere, the PPE-1 promoter is relatively unaffected by diseaseprogression.

EXAMPLE 21

[0298] Assays of the PPE-1 Promoter in a Wound Healing Model

[0299] In order to test the Ad5PPE-1 constructs efficiency and specificactivity in directing Luciferase expression to healing wound bloodvessels, a murine wound healing as described hereinabove in Material andMethods was employed.

[0300] As in other experiments, Ad5CMVLuc was used as a non-tissuespecific control. Luciferase activity under the PPE-1 promoter (FIG. 34;open bars) control was higher both in the normal (6.8±3.2) and inhealing wound region (5±1.6) compared to the activity observed under theCMV control (FIG. 34; black bars).

[0301] Because both the CMV and PPE-1 promoter exhibited reducedexpression levels in the healing wound, these results are difficult tointerpret. Despite this unexpected observation, it is clear that thePPE-1 promoter drives higher levels of expression than the CMV promoterin both normal and healing tissue. The presence of necrotic scar tissuemay account for the reduced expression levels observed with bothpromoters in the healing wound.

[0302] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by their accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence identified by theiraccession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

1 8 1 1334 DNA Mus musculus 1 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgtgtgtgtgtgt gtgtagtgta cttctgatcg 60 gcgatactag ggagataagg atgtacctgacaaaaccaca ttgttgttgt tatcattatt 120 atttagtttt ccttccttgc taactcctgacggaatcttt ctcacctcaa atgcgaagta 180 ctttagttta gaaaagactt ggtggaaggggtggtggtgg aaaagtaggg tgatcttcca 240 aactaatctg gttccccgcc cgccccagtagctgggattc aagagcgaag agtggggatc 300 gtccccttgt ttgatcagaa agacataaaaggaaaatcaa gtgaacaatg atcagcccca 360 cctccacccc acccccctgc gcgcgcacaatacaatctat ttaattgtac ttcatacttt 420 tcattccaat ggggtgactt tgcttctggagaaactcttg attcttgaac tctggggctg 480 gcagctagca aaaggggaag cgggctgctgctctctgcag gttctgcagc ggtctctgtc 540 tagtgggtgt tttctttttc ttagccctgcccctggattg tcagacggcg ggcgtctgcc 600 tctgaagtta gccgtgattt cctctagagccgggtcttat ctctggctgc acgttgcctg 660 tgggtgacta atcacacaat aacattgtttagggctggaa taaagtcaga gctgtttacc 720 cccactctat aggggttcaa tataaaaaggcggcggagaa ctgtccgagt cagacgcgtt 780 cctgcaccgg cgctgagagc ctgacccggtctgctccgct gtccttgcgc gctgcctccc 840 ggctgcccgc gacgctttcg ccccagtggaagggccactt gctgaggacc gcgctgagat 900 ctaaaaaaaa aacaaaaaac aaaaaacaaaaaaacccaga ggcgatcaga gcgaccagac 960 accgtcctct tcgttttgca ttgagttccatttgcaaccg agttttcttt ttttcctttt 1020 tccccactct tctgacccct ttgcagaatggattattttc ccgtgatctt ctctctgctg 1080 ttcgtgactt tccaaggagc tccagaaacaggtaggcgcc acttgcgaat ctttctactt 1140 cagcgcagca gttatcgctt ctgttttccacttttctttc tttcttttct ttcattcttt 1200 cctttttatt tattttttta attactgaagctccagcagc aagtgcctta caattaatta 1260 acttctgtgt gaagcgaaag aaataaaacccctgtttgaa tacagctgac tacaaccgag 1320 tatcgcatag cttc 1334 2 96 DNAArtificial sequence Synthetic oligonucleotide 2 gctagcgtac ttcatacttttcattccaat ggggtgactt tgcttctgga gggtgacttt 60 gcttctggag ccaatgggtacttcatactt ttcatt 96 3 96 DNA Artificial sequence Syntheticoligonucleotide 3 gctagcctcc agaagcaaag tcaccccatt ggaatgaaaa gtatgaagtacaatgaaaag 60 tatgaagtac ccattggctc cagaagcaaa gtcacc 96 4 6 DNAArtificial sequence Nhe-1 restriction site 4 gctagc 6 5 6 DNA Musmusculus misc_feature Hypoxia responsive element - E-box 5 gcacgt 6 6 44DNA Mus musculus misc_feature Murine endothelial specific enhancerelemet 6 gtacttcata cttttcattc caatggggtg actttgcttc tgga 44 7 143 DNAArtificial sequence A triplicate copy of a murine enhancer sequenceoriginated from the PPE-1 promoter 7 gtacttcata cttttcattc caatggggtgactttgcttc tggagggtga ctttgcttct 60 ggagccagta cttcatactt ttcattgtacttcatacttt tcattccaat ggggtgactt 120 tgcttctgga ggctagctgc cag 143 8 47DNA Artificial sequence EDC fragment 8 ctggagggtg actttgcttc tggagccagtacttcatact tttcatt 47

What is claimed is:
 1. An isolated polynucleotide functional as apromoter in eukaryotic cells, the isolated polynucleotide comprising anenhancer element including at least one copy of the sequence set forthin SEQ ID NO:8.
 2. The isolated polynucleotide of claim 1, wherein saidenhancer element further includes at least one copy of the sequence setforth in SEQ ID NO:6.
 3. The isolated polynucleotide of claim 1, whereinsaid enhancer element includes one copy of the sequence set forth in SEQID NO:8 and at least two copies of the sequence set forth in SEQ IDNO:6.
 4. The isolated polynucleotide of claim 1, further comprising anendothelial specific promoter element.
 5. The isolated polynucleotide ofclaim 4, wherein said endothelial specific promoter element comprises atleast one copy of the PPE-1 promoter.
 6. The isolated polynucleotide ofclaim 1, further comprising a hypoxia response element.
 7. The isolatedpolynucleotide of claim 6, wherein said hypoxia response elementincludes at least one copy of the sequence set forth in SEQ ID NO:
 5. 8.The isolated polynucleotide of claim 1, wherein said enhancer element isas set forth in SEQ ID NO:
 7. 9. A nucleic acid construct comprising theisolated polynucleotide of claim 1 and a nucleic acid sequence ofinterest, said nucleic acid sequence of interest being under regulatorycontrol of said isolated polynucleotide.
 10. The nucleic acid constructof claim 9, wherein said nucleic acid sequence of interest is selectedfrom the group consisting of VEGF, p55 and PDGF-BB.
 11. A mammalian celltransformed with the isolated polynucleotide of claim
 1. 12. A method ofexpressing a nucleic acid sequence of interest in endothelial cells, themethod comprising administering to a subject a construct, said constructcomprising the nucleic acid sequence of interest positioned under theregulatory control of a promoter functional in eukaryotic cells, and anenhancer element including at least one copy of the sequence set forthin SEQ ID NO:8.
 13. The method claim of 12, wherein said promoterexhibits endothelial cell specificity.
 14. The method of claim 13,wherein said promoter is the PPE-1 promoter as set forth in SEQ IDNO:
 1. 15. The method of claim 12, wherein said enhancer element furtherincludes at least one copy of the sequence set forth in SEQ ID NO:6. 16.The method of claim 15, wherein said enhancer element is as set forth inSEQ ID NO:
 7. 17. The method of claim 15, wherein said at least one copyof the sequence set forth in SEQ ID NO:6 includes two copies.
 18. Themethod of claim 15, wherein said at least two copies of the sequence setforth in SEQ ID NO:6 are contiguous.
 19. The method of claim 12, whereinsaid nucleic acid sequence of interest is selected from the groupconsisting of VEGF, p55 and PDGF-BB.
 20. The method of claim 12, whereinadministering is effected by a method selected from the group consistingof: (i) systemic in-vivo administration; (ii) ex-vivo administration tocells removed from a body of a subject and subsequent reintroduction ofsaid cells into said body of said subject; and (iii) local in-vivoadministration.
 21. A method of regulating angiogenesis in a tissue, themethod comprising administering a nucleic acid construct including: (a)an endothelial cell specific promoter; (b) at least one copy of ahypoxia response element set forth in SEQ ID NO:5; and (c) a nucleicacid sequence encoding an angiogenesis regulator, said nucleic acidsequence being under regulatory control of said promoter and saidhypoxia response element.
 22. The method of claim 21, whereinadministering is effected by a method selected from the group consistingof: (i) systemic in-vivo administration; (ii) ex-vivo administration tocells removed from a body of a subject, said cells subsequentlyreintroduced into said body of said subject; and (iii) local in-vivoadministration.
 23. The method of claim 21, wherein said nucleic acidconstruct further includes: (d) an enhancer element including at leastone copy of the sequence set forth in SEQ ID NO:8.
 24. The method ofclaim 23, wherein said enhancer element is as set forth in SEQ ID NO: 7.25. The method of claim 23, wherein said nucleic acid construct furtherincludes; (e) at least one copy of a sequence set forth in SEQ ID NO: 6.26. The method of claim 24, wherein said at least one copy of a sequenceset forth in SEQ ID NO: 6 includes two contiguous copies.
 27. The methodof claim 21, wherein said endothelial cell specific promoter comprisesat least one copy of the PPE-1 promoter.
 28. The method of claim 21,wherein said nucleic acid sequence encoding an angiogenesis regulator isselected from the group consisting of VEGF, p55 and PDGF-BB.
 29. Anisolated polynucleotide functional as a promoter in eukaryotic cells,the isolated polynucleotide comprising an enhancer element including thesequence set forth in SEQ ID NO:
 7. 30. The isolated polynucleotide ofclaim 29, further comprising an endothelial specific promoter element.31. The isolated polynucleotide of claim 30, wherein said endothelialspecific promoter element comprises at least one copy of the PPE-1promoter.
 32. The isolated polynucleotide of claim 29, furthercomprising a hypoxia response element.
 33. The isolated polynucleotideof claim 32, wherein said hypoxia response element includes at least onecopy of the sequence set forth in SEQ ID NO:
 5. 34. A nucleic acidconstruct comprising the isolated polynucleotide of claim 29 and anucleic acid sequence of interest, said nucleic acid sequence ofinterest being under regulatory control of said isolated polynucleotide.35. The nucleic acid construct of claim 34, wherein said nucleic acidsequence of interest is selected from the group consisting of VEGF, p55and PDGF-BB.
 36. A mammalian cell transformed with the isolatedpolynucleotide of claim
 29. 37. A method of regulating angiogenesis in atissue, the method comprising administering a nucleic acid constructincluding: (a) an endothelial cell specific promoter; (b) an enhancerelement including the sequence set forth in SEQ ID NO:
 7. (c) at leastone copy of a hypoxia response element set forth in SEQ ID NO:5; and (d)a nucleic acid sequence encoding an angiogenesis regulator, said nucleicacid sequence being under regulatory control of said promoter, saidenhancer element and said hypoxia response element.
 38. The method ofclaim 37, wherein administering is effected by a method selected fromthe group consisting of: (i) systemic in-vivo administration; (ii)ex-vivo administration to cells removed from a body of a subject, andsubsequent reintroduction of said cells into said body of said subject;and (iii) local in-vivo administration.
 39. The method of claim 37,wherein said endothelial cell specific promoter comprises at least onecopy of the PPE-1 promoter (SEQ ID NO: 1).
 40. The method of claim 37,wherein said nucleic acid sequence encoding an angiogenesis regulator isselected from the group consisting of VEGF, p55 and PDGF-BB.
 41. Amethod of regulating angiogenesis in a tissue, the method comprisingadministering a nucleic acid construct including: (a) an endothelialcell specific promoter; (b) an enhancer element including at least onecopy of the sequence set forth in SEQ ID NO:8; and (c) a nucleic acidsequence encoding an angiogenesis regulator, said nucleic acid sequencebeing under regulatory control of said promoter and said enhancerelement.
 42. The method of claim 41, wherein said enhancer elementfurther includes at least one copy of the sequence set forth in SEQ IDNO:6.
 43. The method of claim 41, wherein said enhancer element includesone copy of the sequence set forth in SEQ ID NO:8 and at least twocopies of the sequence set forth in SEQ ID NO:6.
 44. The method of claim41, wherein the a nucleic acid construct further includes a hypoxiaresponse element.